Polysaccharides from Metarhizium pinghaense: a novel biomaterial to modulate bovine satellite cell fate for cultured meat

Polysaccharides from Metarhizium pinghaense: a novel biomaterial to modulate bovine satellite cell fate for cultured meat | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Polysaccharides from Metarhizium pinghaense: a novel biomaterial to modulate bovine satellite cell fate for cultured meat Ji Hoon Park, Ji Won Jang, Si Won Jang, Ye Rim Kim, Jae Ho Han, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7935981/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract The finite proliferative lifespan and progressive loss of stemness in muscle stem cells present a significant bottleneck for the industrial-scale manufacturing of cultured meat. To overcome this limitation, we evaluated a microbial polysaccharide from Metarhizium pinghaense 15R (MP15R) for its capacity to augment bovine satellite cell (BSC) functionality. Our in vitro findings demonstrate that MP15R treatment enhanced BSC proliferation and migration while sustaining elevated expression of the canonical stem cell marker PAX7 throughout prolonged passaging. Moreover, upon induction, MP15R robustly promoted terminal myogenic differentiation, evidenced by the upregulation of key markers such as MYOG and MYHC and an increased myotube fusion index. Collectively, these results establish the MP15R polysaccharide as a sustainable, cost-effective bioactive agent with a bifunctional capacity to improve both the expansion and differentiation phases essential for cultured meat bioprocessing. Biological sciences/Biotechnology Biological sciences/Cell biology Biological sciences/Stem cells Cultured meat Bovine muscle stem cells Polysaccharides Cell proliferation Myogenic differentiation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION With the rise in global meat consumption, concern over the environmental impact and sustainability of traditional livestock farming is increasing. Therefore, cultured meat has emerged as a promising alternative that offers potential benefits in terms of resource efficiency, animal welfare, and greenhouse gas reduction.( 1 , 2 ) Efficient large-scale production of muscle tissue is essential in the cultured meat industry because muscle is not only the primary structural and nutritional component of meat but also a key factor in reducing the overall manufacturing costs and meeting consumer expectations.( 3 – 5 ) As a protein-dense and nutritionally vital component, muscle is central to the nutritional value of cultured meat, and its mass production is essential for meeting both the nutritional requirements of the growing population and consumer expectations.( 6 , 7 ) Satellite cells are muscle stem cells located between the basal lamina and sarcolemma, and they play a key role in muscle regeneration and maintenance. These cells remain in a quiescent state and become activated in response to muscle injury or other physiological signals.( 8 – 10 ) Upon activation, satellite cells proliferate and differentiate to form new muscle fibers or fuse with existing fibers to maintain and repair muscle tissue.( 11 ) Satellite cell functions are regulated by transcription factors, such as paired box 7 (PAX7), and their proliferation and differentiation are significantly influenced by signaling pathways, such as Notch and Wnt.( 12 , 13 ) In addition, satellite cell activation and differentiation are modulated by various external cues, including inflammatory responses, growth factors, and mechanical stimuli. Dysregulation of these processes can lead to muscle atrophy and muscle-related diseases.( 14 ) Muscle stem cells proliferate for a certain period of time and then stop proliferating, or their function deteriorates with cell aging.( 15 , 16 ) Appropriate culture medium and nutrients are required to promote the proliferation of muscle stem cells. However, a clear mechanism for cell proliferation has not yet been fully elucidated, and continuous improvements in culture conditions are needed.( 17 , 18 ) Recent studies have attempted to overcome the challenges of muscle stem cell proliferation by treatment with small molecules or optimization of culture media composition. For instance, the inhibition of p38 mitogen-activated protein kinase using small molecules such as SB203580 has been shown to enhance the self-renewal capacity of muscle stem cells and maintain their regenerative potential during ex vivo expansion.( 19 ) Additionally, receptor tyrosine kinase inhibitors, such as CEP-701, can promote satellite cell expansion and improve muscle regeneration.( 20 ) However, the large-scale production of muscle stem cells remains a significant challenge. Traditional culture methods often lead to the spontaneous differentiation or senescence of muscle stem cells, limiting their proliferative capacity and therapeutic potential.( 21 – 23 ) Polysaccharides have attracted attention as potential alternatives for overcoming these limitations owing to their broad spectrum of biological activities. They are naturally occurring polymers found in a wide range of organisms, including plants, animals, algae, fungi, and bacteria.( 24 ) In particular, microbially derived polysaccharides can be produced in large quantities within a short time period, making them highly cost-effective and a subject of active research.( 25 ) Moreover, because of their diverse physiological activities, natural polysaccharides have the potential to improve cellular functions such as alleviating colitis and upregulating immune cells derived from mouse bone marrow.( 26 – 28 ) For example, crude polysaccharides extracted from Ecklonia cava have been reported to enhance the proliferation and differentiation of Hanwoo muscle satellite cells by activating pathways such as hepatocyte growth factor, its receptor MET, and focal adhesion kinase.( 29 , 30 ) Entomopathogenic fungi are a group of microorganisms that infect and ultimately kill insects by attaching to the insect cuticle, penetrating the host body, and causing death within a few days.( 31 ) Among the approximately 80,000 known fungal species, only approximately 700 are entomopathogenic and exhibit a unique life cycle that involves parasitizing and killing insect hosts. These fungi are considered harmless to humans and characterized by high environmental stability and host specificity, making them promising candidates as biological control agents to replace conventional chemical pesticides.( 32 – 34 ) Some entomopathogenic fungi belonging to the family Cordycipitacege, including Cordyceps, form fruiting bodies after killing their insect hosts. These Cordyceps possess high pharmacological value and exhibit anti-inflammatory, antioxidant, hypoglycemic, and immunomodulatory activities; thus, have been widely used in traditional medicine.( 32 ) Owing to these beneficial properties, entomopathogenic fungi and Cordyceps have been actively studied and developed as pharmaceutical and nutraceutical agents.( 32 , 33 ) In addition to their secondary metabolites, such as cordycepin and destruxin,( 35 , 36 ) which are known for their pharmacological effects, entomopathogenic fungi also produce large amounts of bioactive polysaccharides with significant therapeutic potential. Polysaccharides derived from Metarhizium taii have been reported to induce insulin resistance in HepG2 cells,( 37 ) whereas those from Cordyceps militaris exhibit antitumor and immune-enhancing effects.( 38 , 39 ) Although these findings indicate the biological and pharmacological potential of entomopathogenic fungal polysaccharides, their applicability in cell culture systems remains poorly understood. In particular, the use of these polysaccharides in muscle stem cells has not yet been thoroughly investigated. Although preliminary studies suggest potential benefits, comprehensive research is required to elucidate the mechanisms underlying the influence of polysaccharides on muscle stem cell behavior and assess their efficacy in large-scale cell culture systems. In the present study, we investigated the effects of polysaccharides derived from entomopathogenic fungi on the proliferation and differentiation of bovine satellite cells (BSCs). For this purpose, we employed two fungal strains from Tolypocladium cylindrosporum (T15 and T237) and one strain from Metarhizium pinghaense 15R (MP15R), which were previously observed to produce high levels of polysaccharides. The results demonstrated that the polysaccharides derived from these entomopathogenic fungi significantly enhanced BSCs proliferation and promoted the expression of muscle differentiation markers, such as myosin heavy chain (MYHC) and myogenin (MYOG), thereby facilitating the proliferation and differentiation of BSCs. These findings suggest novel strategies for harnessing their potential for cultured meat production and regenerative medicine. RESULTS Screening of microbial polysaccharides identifies MP15R as a potent enhancer of BSCs viability To assess the cytotoxicity of the polysaccharides, the BSCs were treated with the polysaccharides at various concentrations (0.05–20 µg/mL), and then CCK-8 assays were performed (Fig. 1 A, 1 B, 1 C). All experiments were performed in triplicate (n = 3) and statistical analyses were performed using Duncan’s multiple comparison test (p < 0.05). The results revealed that cell viability in the 0.05 µg/mL, 10 µg/mL, 5 µg/mL, and 20 µg/mL MP15R polysaccharide groups was higher than that of the NC. The highest cell viability was observed at a concentration of 5 µg/mL, which was approximately 23.8% higher than that of the NC group (Fig. 1 A) (p < 0.001). In the polysaccharide T15 treatment group, the highest cell viability was observed at 500 ng/mL, which was similar to that of the NC group. However, when treated with a high concentration of 500 ng/mL or higher, cell viability tended to gradually decrease (Fig. 1 B) (p < 0.001). Finally, in the polysaccharide T237 treatment group, the highest cell viability was observed at a concentration of 100 ng/mL, although the cell viability was approximately 10% lower than that of the NC group. When treated with a high concentration of 100 ng/mL or higher, the cell viability tended to gradually decrease (Fig. 1 C) (p < 0.001). Based on these results, MP15R polysaccharide at 5 µg/mL was selected for subsequent experiments due to its superior effect on BSC proliferation. MP15R polysaccharide promotes long-term proliferation and enhances the migratory capacity of BSCs Based on the previous screening results, MP15R polysaccharide at a concentration of 5 µg/mL was identified as the most effective condition for promoting BSC proliferation and thus was selected for further evaluation of its long-term effects. First, to assess these effects, the morphological characteristics and cell proliferation were evaluated at passages 3, 5, 7, and 9. Morphological evaluation showed no obvious visual differences between the NC and MP15R polysaccharide-treated groups (Fig. 2 A). Under both conditions, the cells exhibited a typical spindle-shaped morphology, and the cell density gradually decreased with passage. Cell proliferation, as assessed by the cell count, was consistently higher in the treated group than in the NC group (Fig. 2 B). This difference was statistically significant at passage 7 (p < 0.05), suggesting a potential growth-promoting effect of the MP15R polysaccharide at this stage. Second, since cell migration plays a key role in biological processes such as muscle differentiation, development, and tissue repair, a wound-healing assay was conducted to evaluate the effect of the MP15R polysaccharide on the migration of BSCs.( 40 ) The assay was performed at passage 3 to determine whether the additive influenced the migratory capacity of the cells at an early stage. Morphologically, the wounds appeared narrower in the MP15R polysaccharide-treated group (5 µg/mL) compared to the NC (Fig. 2 C). To quantify this, the wound area was measured using ImageJ (version 1.54g, National Institutes of Health, USA). A comparison of scratch distances between 0 and 24 h revealed a migration index of 49.26% in the NC group and 60.13% in the MP15R polysaccharide-treated group, with the difference being statistically significant (p < 0.01) (Fig. 2 D). This enhanced migration suggests a possible regulatory role of the MP15R polysaccharide in the proliferation and differentiation of BSCs. MP15R treatment accelerates cell cycle progression by promoting S-phase entry Analysis of the cell cycle phase distribution of the BSC population is a direct method of measuring how many cells are activated and how many are dormant.( 41 , 42 ) To assess the impact of the MP15R polysaccharide on the proliferative capacity of BSCs, a cell cycle analysis was conducted. Previous studies have demonstrated that alterations in the extracellular matrix or signaling pathways can influence the distribution of BSCs across different cell cycle phases, thereby affecting their proliferation and differentiation potential. The cell cycle analysis was performed at passages 3 and 5. In the G0/G1 phase, both p3 and p5 were slightly higher in the treatment group (5 µg/mL) than in the NC group, with a significant difference between the p3 NC groups (p < 0.05). The S phase was 4.3% and 3.9% in the p3 and p5 NC groups, while it was 7.3% and 5.6% in the p3 and p5 treatment groups (5 µg/mL). The S phase gradually decreased with the passage, but the population of the treatment group (5 µg/mL) was significantly higher than that of the NC group (p3, p < 0.001; p5, p < 0.05). The G2/M phase naturally decreased as the G0/G1 and S phases increased, suggesting that the overall proliferation activity was enhanced by rapid cycle rotation (p3, p < 0.01; p5, p < 0.01) (Fig. 3 A, 3 B). Our results indicate that treatment with the MP15R polysaccharide can influence the proliferation and cell cycle dynamics of BSCs during passages 3 and 5, suggesting its potential impact on both short- and long-term cellular behavior. MP15R polysaccharide preserves the undifferentiated state of BSCs during extended culture The expression patterns of PAX7 and MYOD1 in BSCs were examined using immunocytochemistry (ICC) and quantitative PCR (qPCR). ICC showed positive staining for both markers in NC and MP15R polysaccharide-treated groups at passage. Although both proteins were visualized by double immunostaining, only PAX7-positive nuclei were quantitatively analyzed (Fig. 4 A). The proportion of PAX7 + cells, expressed as a percentage of the total DAPI-stained nuclei, was higher in the MP15R polysaccharide-treated group than in the NC group, indicating that the MP15R polysaccharide may support the preservation of stemness in satellite cells through the early upregulation of PAX7 (p < 0.05) (Fig. 4 B). qPCR analysis revealed that PAX7 mRNA levels remained consistently higher in the MP15R polysaccharide-treated group throughout passages 3, 5, 7, and 9, with a significant increase observed at passage 3 (PAX7, p3, p < 0.05; MYOD1, p3, p < 0.01). In contrast, MYOD1 expression displayed a more variable pattern, with significantly higher expression in the MP15R polysaccharide-treated group and NC group at passages 3 (p < 0.01) and 7 (p < 0.001), respectively (Fig. 4 C). No significant differences were observed between passages 5 and 9. These findings indicate that MP15R polysaccharides may enhance early myogenic activation, particularly by upregulating PAX7 and MYOD1 at the early stages, while exerting a more limited or passage-dependent effect on MYOD1 expression. MP15R polysaccharide enhances the terminal myogenic differentiation of BSCs The effect of MP15R polysaccharide on BSC differentiation was assessed by adding the polysaccharide to the differentiation medium at various concentrations. Since 5 µg/mL MP15R polysaccharide showed a positive effect on proliferation in the growth medium, an additional experiment was conducted in which cells were first cultured in growth medium containing 5 µg/mL MP15R polysaccharide and then switched to differentiation medium. After four days of differentiation under the five different treatment conditions, MYHC expression and myotube formation were assessed by ICC (Fig. 5 A). MYHC-positive multinucleated myotubes were observed in all groups. However, no obvious morphological differences in myotube formation were observed among the groups, including the NC and MP15R polysaccharide-treated groups. Quantification of the fusion index, calculated as the percentage of DAPI-stained nuclei within MYHC-positive myotubes, revealed statistically significant increases in the GM + 1 µg and 5 µg + DM groups compared to the NC (GM + DM) (p < 0.05) (Fig. 5 B). To examine the effect of the MP15R polysaccharide on gene expression, the mRNA levels of MYHC and MYOG were analyzed by qPCR (Fig. 5 C). Both genes were significantly upregulated in the GM + 1 µg and 5 µg + DM groups relative to the NC. Statistical analysis using Duncan’s multiple range test identified distinct groupings (a-c), thus confirming significant differences in gene expression among the treatment conditions (MYHC, p < 0.05; MYOG, p < 0.0001). These results suggest that the MP15R polysaccharide promotes BSC differentiation at the molecular level, particularly when applied during early differentiation or following pretreatment in a growth medium. DISCUSSION As the demand for scalable and sustainable cultured meat production grows, increasing attention has been focused on biomaterials capable of supporting muscle stem cell expansion.( 1 , 3 ) Natural polysaccharides, known for their bioactivity in mammalian systems, such as immune modulation and tissue regeneration, have been used as supplements in various cell culture contexts.( 29 , 30 ) However, their specific effects on BSCs remain poorly characterized, and to our knowledge, no previous study has investigated the dual role of fungal-derived polysaccharides in promoting both proliferation and differentiation of BSCs under long-term culture conditions. In this study, we examined the effects of a fungal polysaccharide derived from strain MP15R on the proliferation, migration, cell cycle, and differentiation of BSCs. Our results demonstrated that the MP15R polysaccharide significantly improved cell viability in a dose-dependent manner, with 5 µg/mL identified as the optimal concentration (Fig. 1 A). This suggests that treatment with an optimal concentration of the polysaccharide exerts beneficial effects on cell culture. In long-term cultures, BSCs treated with the MP15R polysaccharide exhibited consistently higher cell numbers than those in the NC group, with a statistically significant difference at passage 7 (Fig. 2 B). Thus, the MP15R polysaccharide may support prolonged expansion without early senescence. Increased S-phase populations observed via flow cytometric analysis further confirmed the proliferative effect of the MP15R polysaccharide, particularly in early passages. As shown in Fig. 3 B, a significant increase in the S-phase population was observed at passages 3 and 5 in the MP15R polysaccharide-treated group, suggesting enhanced entry into the cell cycle and elevated DNA synthesis. Notably, the decrease in G2/M phase may have reflected accelerated cell cycle progression, resulting in increased proliferation. Although the S-phase proportion decreased at passage 5 compared to passage 3, this may indicate a passage-dependent decline in responsiveness to the MP15R polysaccharide, potentially due to differential utilization across specific cell cycle phases.( 43 ) Additionally, BSCs cultured with the MP15R polysaccharide demonstrated significantly enhanced migration capacity, as evidenced by a higher migration index in wound-healing assays at 24 h (Fig. 2 D). Enhanced motility may contribute to faster tissue formation and improved responsiveness to regenerative cues in vitro.( 44 ) Gene and protein expression analyses revealed that MP15R polysaccharide treatment increased PAX7 expression in both early and late passages, and ICC confirmed a higher population of PAX7 + nuclei (Fig. 4 A, 4 B). PAX7 is a key transcription factor that maintains the identity and self-renewal ability of BSCs. Its sustained expression suggests that the MP15R polysaccharide preserves the stemness of BSCs during long-term expansion.( 9 , 10 , 45 ) While MYOD1 expression was transiently elevated at early passages (Fig. 4 C), it declined by passage 7 in the treated cells, suggesting a controlled or delayed commitment to differentiation. Interestingly, the fusion index and expression of myogenic differentiation markers (MYHC and MYOG) were significantly upregulated when the MP15R polysaccharide was administered at specific stages during the differentiation protocol. In particular, the 5 µg + DM group (pretreatment in growth media followed by normal differentiation) showed significant increases in both the fusion index and gene expression (Fig. 5 B, 5 C). These results suggest that the MP15R polysaccharide supports not only the proliferation but also the differentiation potential of BSCs when applied at the appropriate time points. Compositional analysis of MP15R polysaccharide revealed that its predominant monosaccharides are galactose (52.11 mg/g), mannose (48.53 mg/g), arabinose (4.16 mg/g), and fucose (2.86 mg/g), while glucose was not detected. Mannose is efficiently metabolized by cultured cerebellar granule neurons,( 46 ) while D-galactose induces cellular senescence in C2C12 myoblasts.( 47 ) However, the specific effects of galactose and mannose on muscle stem cells remain largely unexplored, making it difficult to predict how the monosaccharide composition of the MP15R polysaccharide influences these cells. In addition, the MP15R polysaccharide is characterized as an anionic polysaccharide containing 18.54% sulfate groups;( 48 ) however, the precise mechanisms by which these negatively charged moieties affect muscle stem cells remain unclear. Sulfated polysaccharides are known to possess strong immunomodulatory potential because they regulate macrophages and natural killer (NK) cells and promote homeostasis by enhancing cytokine secretion and immune function.( 49 – 51 ) Based on these immunological properties, it is plausible that the MP15R polysaccharide exerts beneficial effects on muscle cell growth and immune regulation. Therefore, the observed enhancement in cell proliferation is likely attributable to the combined effects of various saccharide residues and sulfate groups present in the MP15R polysaccharide rather than the action of any single monosaccharide component alone.( 52 ) From an applied perspective, this study demonstrated that the MP15R polysaccharide, previously considered a byproduct of the cultivation of entomopathogenic fungi, exhibits meaningful biological activity by promoting cell cycle progression and accelerating the recovery of damaged cells. These findings suggest the potential use of the MP15R polysaccharide as a culture medium supplement to support the growth and differentiation of BSCs. Notably, this polysaccharide holds promise as a novel growth-promoting biomaterial that can overcome the limitations associated with conventional chemical growth factors, such as cost and stability. Furthermore, our findings suggest that it offers a promising strategy for overcoming a major bottleneck in cultured meat bioprocessing by maintaining satellite cell populations that are both expandable and myogenically competent. Traditional culture methods often induce spontaneous differentiation or reduce the regenerative potential of muscle stem cells over time.( 21 , 22 ) Our findings indicate that the MP15R polysaccharide may function as a biomaterial additive to address these limitations and improve biomass production from BSCs. This study demonstrates that polysaccharides derived from entomopathogenic fungi can significantly enhance the proliferation, migration, and myogenic differentiation of BSCs in vitro. By promoting PAX7 expression and S-phase entry while maintaining stem-like characteristics over multiple passages, the MP15R polysaccharide contributes to the long-term expansion of functional muscle stem cells. In addition, the increased expression of MYHC and MYOG, along with the improved fusion indices under specific treatment conditions, suggests that the MP15R polysaccharide not only preserves the regenerative capacity of BSCs but also primes the cells for efficient differentiation when required (Fig. 5 B, 5 C). These findings hold substantial significance for the cultured meat industry, where one of the key challenges lies in maintaining a scalable and renewable source of high-quality muscle cells. The ability of the MP15R polysaccharide to simultaneously support stemness and differentiation under controlled conditions makes it a valuable candidate for developing improved bioprocessing strategies. Furthermore, the use of natural, microbial-derived polysaccharides aligns with the principles of sustainability and cost efficiency, which are critical for the transition from laboratory-scale to industrial-scale cultured meat production.( 53 ) Although further studies are required to elucidate the underlying molecular mechanisms and assess their performance in 3D culture systems or bioreactors, our results lay the groundwork for future exploration of polysaccharide-based supplements in muscle stem cell culture. Overall, this study provides novel insights into how functional biomolecules from microbial sources can be repurposed to enhance muscle cell performance and paves the way for innovative approaches in regenerative biology and cell-based meat engineering. Despite the promising findings, this study has several limitations that should be considered. First, the effects of the MP15R polysaccharide were evaluated exclusively under 2D monolayer conditions. While this approach is suitable for initial screening, it does not fully recapitulate the complex three-dimensional environment of native muscle tissue or the dynamic conditions within an industrial bioreactor. Therefore, future investigations are necessary to validate whether similar benefits are observed in 3D culture systems, such as scaffolds or microcarriers, which would be critical for assessing its practical applicability. Second, while this study characterized the phenotypic outcomes of MP15R treatment, the precise molecular mechanisms underlying its bioactivity were not elucidated. Although the precise molecular mechanisms by which the MP15R polysaccharide acts remain unclear, previous studies have demonstrated that polysaccharides can modulate intracellular signaling pathways, such as mitogen-activated protein kinase and PI3K/AKT, both of which are closely linked to satellite cell proliferation and differentiation.( 30 , 54 , 55 ) A comprehensive mechanistic investigation is required to fully understand how MP15R interacts with cellular machinery to regulate cell fate. Clarifying these pathways will be essential for optimizing its application and for the rational design of next-generation biomaterials for cultured meat bioprocessing. In conclusion, this study demonstrated that the MP15R polysaccharide promotes BSC proliferation during long-term culture and enhances muscle differentiation efficiency at specific concentrations. Polysaccharides generally have low cytotoxicity and high biocompatibility, which allow them to maintain cell viability and function during long-term culture. These properties suggest their potential as practical supplements for stably supplying the cells required for cultured meat production. Materials and Methods Fungal strain and polysaccharides extraction The entomopathogenic fungus Metarhizium pinghaense 15R (KACC 83065BP) was obtained from the Korean Agricultural Culture Collection (KACC, Korea) and cultured by the Center for Industrialization of Agricultural and Livestock Microorganisms (CIALM, Korea). The dried polysaccharide extract derived from the culture broth was subsequently provided to our laboratory and used for experimental analyses. Tolypocladium cylindrosporum strains T15 and T237 were isolated from sedimentary soils collected from Danyang-gun, Chungcheongbuk-do, and Uljin-gun, Gyeongsangbuk-do, respectively. These strains were stored at − 20°C and, for experimental purposes, cultured in Sabouraud dextrose broth (SDB) (238230; BD DIFCO) medium at 25 ± 2°C with shaking at 150 rpm for 14 days. The culture broths were centrifuged at 10,000 rpm for 20 min at 4°C to remove the mycelial biomass, followed by filtration through filter paper. The resulting supernatants were collected and polysaccharides were precipitated by adding cold ethanol at three times the volume of the supernatant. The precipitates were recovered by centrifugation under the same conditions, and the residual ethanol was removed. All polysaccharides were treated using the Sevag method( 56 ) to remove proteins and lipids. The resulting supernatants were further purified using a 0.45 µm syringe filter (SP25P045NL; Hyundai Micro). Cold absolute ethanol (five volumes) was then added, and the solution was incubated at 4°C for 24 h. After ethanol removal, the precipitated polysaccharides were redissolved in distilled water and filtered through a 0.22 µm syringe filter (SP25P020NL; Hyundai Micro). Ethics Approval This study follows the recommendations of the ARRIVE guidelines. All animal procedures were approved by the Animal Ethics Committee of Jeonbuk National University (JBNU; NON2023-137), Republic of Korea. All experiments were performed in accordance with the ethical guidelines and regulations of the Jeonbuk National University. Bovine satellite cell isolation Bovine satellite cells were isolated from the longissimus dorsi (loin) muscle of a 1-month-old male Korean native calf according to a previously reported protocol ( 57 ). Briefly, 5 g of muscle tissue was weighed and finely chopped using surgical scissors for approximately 5 min. The minced tissue was enzymatically digested for 2 h at 37°C in a solution composed of DMEM/F12 (Gibco, Carlsbad, CA, USA, #11320-033), 0.25% trypsin-EDTA (TE) (Gibco, #25200-072), collagenase II (Worthington, Lakewood, NJ, USA, #CLS-2, 5 g), Dispase II (Roche, Indianapolis, IN, USA, #4942078001, 1 U/mL), and 10% antibiotic-antimycotic (A.A., Gibco, #15240062). After digestion, the cell suspension was neutralized using DMEM low glucose (Gibco, #11885092) supplemented with 15% fetal bovine serum (FBS) (Gibco, 16000-044, 26140079) and 1% A.A. and then centrifuged at 80 × g for 3 min at 4°C. The supernatant was sequentially filtered through 100 µm and 40 µm strainers. Red blood cells were removed using Red Blood Cell (RBC) lysing buffer (Sigma-Aldrich, St. Louis, MO, USA, #R7757-100mL) and then washed twice with PBS. The remaining cells were resuspended in primary culture media containing Ham’s F-10 (Gibco, #11550-043), 20% FBS, 1% A.A., basic fibroblast growth factor (bFGF) (R&D System, Minneapolis, MN, USA, #223-FB-500/CF, 5 ng/mL), and Primocin (Invivogene, Pak Shek Kok, New Territories, Hong Kong, ant-pm-2, 100 µg/mL) until reaching a cell density 70–80% confluence. Before use, the medium was switched to a formulation without Primocin, and penicillin-streptomycin (PS) was replaced with A.A. The experiment was performed from passages 3 to 9 to evaluate how the polysaccharide treatment influenced the proliferation and maintenance of BSCs over a long-term culture. Satellite cell culture and differentiation Primary muscle-derived cells (passage 2) were maintained on culture dishes that had been pre-coated with a 0.1% gelatin solution (G1319; Sigma-Aldrich, St. Louis, MO, USA). For standard expansion, bovine satellite cells (BSCs) were cultured in a growth medium composed of Ham’s F-10 (Gibco, #11550-043) supplemented with 20% FBS, 1% Antibiotic-Antimycotic, and 5 ng/mL bFGF. In the experimental groups, the medium was additionally supplemented with the designated polysaccharides during each feeding. The cells were subcultured every four days, with the culture medium being refreshed every two days. To assess long-term proliferation, BSCs were plated onto 6-well plates at a density of 4.8 × 10⁴ cells per well. At each subculture from passage 3 (P3) to P9, cells were harvested, and total cell counts for each group were determined in triplicate using a LUNA-FL™ Dual Fluorescence Cell Counter (Logos Biosystems, Cat# L20001). The seeding densities for other experimental formats were as follows: 1 × 10⁶ cells per 100 mm dish, 1 × 10⁴ cells per well for 4-well plates, and 6.4 × 10² cells per well for 96-well plates. To induce myogenic differentiation, BSCs were initially expanded in the growth medium as described. For these experiments, cells were seeded at 9.6 × 10⁴ cells per well in 6-well plates or 2 × 10⁴ cells per well in 4-well plates. Following an initial 48-hour proliferation period, the culture medium was switched to a differentiation medium (DM), which consisted of DMEM low glucose, 2% Horse Serum, and 1% Antibiotic-Antimycotic. The cells were then maintained in this DM on gelatin-coated dishes for an additional two days to promote differentiation. Cell proliferation analysis Bovine satellite cells were seeded in a 96-well plate with various concentrations of polysaccharide mixes (MP15R, T15, and T237). The 96-well plate was coated with 0.1% gelatin, and BSCs were seeded in every well at a density of 6.4 × 102 cells. The cells were cultured for 3 days, and cell growth was determined using Cell Counting Kit-8 (CCK-8) (#CK04-11; Dojindo, Kumamoto, Japan). Cells were treated with CCK-8 solution according to the manufacturer’s instructions and incubated at 37°C for 3 h. Growth was measured using a microplate reader at a wavelength of 450 nm. Gene expression analysis by qRT-PCR RNA was extracted from cells using the AccuPrep® Universal RNA Extraction kit (Bioneer, Seoul, Korea) according to the manufacturer’s instruction. One microgram of total RNA from each sample was reverse transcribed to cDNA using the Accupower® CycleScript RT Premix (Bioneer, Seoul, Republic of Korea) according to the manufacturer’s instruction. Relative gene expression was measured in triplicate using Powerup SYBR Green Master Mix (Applied Biosystems, Waltham, MA, USA). The primer sequences are listed in Table S1 . For data normalization, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was employed as the housekeeping gene. Quantitative PCR was performed using a LightCycler® 96 system, and the relative expression levels were determined by the 2⁻ΔCt method.( 58 ) Immunofluorescence staining Cultured BSCs were seeded in a 4-well plate at a density of 1 × 104 and cultured for four days. After four days, the cells were washed twice with PBS and then fixed with cold 4% paraformaldehyde for 20 min at room temperature. The washing solution was composed of PBS containing 0.3% Triton X-100 (PBST), and the blocking solution was PBST containing 3% bovine serum albumin (BSA; Bovogen, Keilor East, Australia). After fixation, the cells were washed three times with PBS and then incubated in blocking solution for 1 h at room temperature for permeabilization and blocking. After blocking, the cells were washed three times with washing solution. After fixation, cells were then stained overnight at 4°C with the following primary antibodies: anti-MYOD1 (Polyclonal, 1:200, Proteintech, Rosemont, IL, USA), anti-PAX7 (PAX7, monoclonal, 1:50, DHSB, Iowa, IA, USA) and anti-MYHC (MF20, monoclonal, 1:20, Iowa City, IA, USA). Subsequently, cells were then washed three times with washing solution stained with Alexa488 anti-mouse (Invitrogen, USA, A11001) antibodies and Alexa568 labeled anti-rabbit (Invitrogen, USA, A11011) antibodies at room temperature for 2 h and stained with 1 µg/mL of 4’,6-diamidino-2-phenylindole (DAPI) for 5 min. After DAPI staining, the cells were washed three times with the washing solution for 10 min. Bovine satellite cells stained with PAX7, MYOD1, and MF20 were captured using a Leica DFC 9000 (Deerfield, IL, USA) at 200x magnification. The expression of PAX7 and MYOD1 was calculated as a ratio of the total DAPI count. Cell cycle analysis Bovine satellite cells were collected during passages 3 and 5 and seeded in a 35 mm dish at a density of 4.8 × 104. For cell cycle analysis, the cells were detached using 0.25% trypsin-EDTA and neutralized media containing DMEM low glucose, 15% FBS, and 1% A.A. Cells were then washed with cold PBS (containing 1% BSA), fixed with 70% ethanol for 5 min at 4°C, and then stored at − 20°C until the day of cell cycle analysis (1–2 weeks). On the day of cell cycle analysis, cells were centrifuged at 850 × g at 4°C for 5 min. The ethanol was removed, and the cells were washed twice with PBS. Afterwards, 100 µg/mL of RNase A (Sigma-Aldrich, #70856) and 25 µg/mL of propidium iodide (PI) (Bio Legend, San Diego, CA, USA, #421301) were added with PBS. The cells were analyzed by flow cytometry using a blue laser (excitation at 488 nm). PAX7 + cell population and fusion index percentage measurement PAX7 + cell populations were quantified by manually counting the number of nuclei costained with DAPI and PAX7 in three randomly selected fields per condition. Percentages were calculated by dividing the number of PAX7 + nuclei by the total number of DAPI-stained nuclei in the same field. The cells were seeded in 4-well plates at a density of 1 × 104 cells/well. Counting was performed manually using PowerPoint-marked fluorescence images. The fusion index was defined as the percentage of nuclei located within the myotubes, which are defined as multinucleated cells containing three or more nuclei, relative to the total number of nuclei in the same field. Cells were seeded at a density of 2 × 104 cells/well and grown in GM for 2 days, followed by the induction of differentiation. Three or more randomly selected fields per condition were analyzed using Myotube Analyzer software (available at https://github.com/SimonNoe/myotube-analyzer-app).(59) Cell migration effect (wound-healing assay) A wound-healing assay was performed to assess cell migration. BSCs were seeded in 6-well plates at a density of 4.8 × 104 cells/well and cultured in growth medium for two days until they reached confluence. A linear scratch was made in each well using a sterile 1000 µL pipette tip, after which the medium was replaced with fresh growth medium to remove detached cells. Images of the scratch areas were captured at 0, 12, and 24 h using a Leica DFC9000 imaging system (Leica Microsystems, Germany) at 50× magnification. Cell migration was quantified by measuring wound closure over time, and the migration index was analyzed using the ImageJ software (NIH, Bethesda, MD, USA). STATISTICAL ANALYSIS P All experiments were performed three times, and the data from all repetitions of each experiment were collated and expressed as the means ± standard error (SE) of the mean. Statistical tests were conducted using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA), and statistical differences were analyzed using Student’s t-test or analysis of variance (ANOVA) followed by Duncan’s multiple range test for post hoc comparisons. Statistical significance was set at p < 0.05. DAta availibility The polysaccharides MP15R, T15, and T237 generated in this study are available from the corresponding author (Hyun Woo Choi, [email protected] ) with a completed material transfer agreement. Any other data supporting the findings of this study are available from the corresponding author upon reasonable request. code availibility This article does not report original code. Declarations Competing interests Several authors (Tae-Young Shin, Hyun-Woo Choi, Ji-Hoon Park and Ji-Won Jang) are inventors on a pending Korean patent application (Application No. 10-2025-0069520) related to the use of Metarhizium pinghaense polysaccharides for modulating bovine satellite cell fate described in this manuscript. The patent application is assigned to Jeonbuk National University Industry-Academic Cooperation Foundation, Chungbuk National University Industry-Academic Cooperation Foundation, and the Center for Industrialization of Agricultural and Livestock Microorganisms (CIAM). The other authors declare no competing interests. supplementaRY information Supplementary Table S1 , listing primer sequences used for qPCR, is available in the online version of this article. Funding This research was supported by the ‘High Value-added Food Technology Development Program’ of the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET), funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA), Republic of Korea (Grant No. 322006-05-CG000). Author Contribution Conceptualization, T.Y.S. and H.W.C.; methodology, J.H.P., and J.W.J.; Investigation, J.H.P., and J.W.J.; writing—original draft, J.H.P. and J.W.J.; writing—review & editing, J.H.P., J.W.J., T.Y.S., and H.W.C.; funding acquisition, T.Y.S. and H.W.C.; resources, J.H.P., J.W.J., S.W.J., Y.R.K., J.H.H., and G.R.N.; supervision, T.Y.S., and H.W.C. All authors have read and agreed to the published version of the manuscript. Data Availability The polysaccharides MP15R, T15, and T237 generated in this study are available from the corresponding author (Hyun Woo Choi, [email protected] ) with a completed material transfer agreement. Any other data supporting the findings of this study are available from the corresponding author upon reasonable request. References Post, M. J. Cultured meat from stem cells: Challenges and prospects. Meat Sci. 92 (3), 297–301 (2012). Ogawa, M. et al. Edible mycelium as proliferation and differentiation support for anchorage-dependent animal cells in cultivated meat production. npj Sci. Food . 8 (1), 23 (2024). Garrison, G. L., Biermacher, J. T. & Brorsen, B. W. How much will large-scale production of cell-cultured meat cost? J. Agric. Food Res. 10 , 100358 (2022). Stout, A. J. et al. Engineered autocrine signaling eliminates muscle cell FGF2 requirements for cultured meat production. bioRxiv (2023). Pasitka, L. et al. Empirical economic analysis shows cost-effective continuous manufacturing of cultivated chicken using animal-free medium. Nat. Food . 5 (8), 693–702 (2024). Guan, X., Zhou, J., Du, G. & Chen, J. Bioprocessing technology of muscle stem cells: implications for cultured meat. Trends Biotechnol. 40 (6), 721–734 (2022). Lim, P. Y., Suntornnond, R. & Choudhury, D. The nutritional paradigm of cultivated meat: Bridging science and sustainability. Trends Food Sci. Technol. 156 , 104838 (2025). Yin, H., Price, F. & Rudnicki, M. A. Satellite Cells and the Muscle Stem Cell Niche. Physiol. Rev. 93 (1), 23–67 (2013). Dumont, N. A., Wang, Y. X. & Rudnicki, M. A. Intrinsic and extrinsic mechanisms regulating satellite cell function. Development 142 (9), 1572–1581 (2015). Hung, M., Lo, H-F., Jones, G. E. L. & Krauss, R. S. The muscle stem cell niche at a glance. J. Cell Sci. ; 136 (24). (2023). ChargÉ, S. B. P. & Rudnicki, M. A. Cellular and Molecular Regulation of Muscle Regeneration. Physiol. Rev. 84 (1), 209–238 (2004). Tsivitse, S. Notch and Wnt Signaling, Physiological Stimuli and Postnatal Myogenesis. Int. J. Biol. Sci. 6 (3), 268–281 (2010). Pasut, A. et al. Notch Signaling Rescues Loss of Satellite Cells Lacking Pax7 and Promotes Brown Adipogenic Differentiation. Cell. Rep. 16 (2), 333–343 (2016). Fukada, S. Exercise/Resistance Training and Muscle Stem Cells. enm 36 (4), 737–744 (2021). Sousa-Victor, P., García-Prat, L., Serrano, A. L., Perdiguero, E. & Muñoz-Cánoves, P. Muscle stem cell aging: regulation and rejuvenation. Trends Endocrinol. Metabolism . 26 (6), 287–296 (2015). Qin, G. et al. Notch signaling modulation enhances porcine muscle stem cell proliferation and differentiation. Biochem. Biophys. Res. Commun. 752 , 151456 (2025). Kolkmann, A. M., Van Essen, A., Post, M. J. & Moutsatsou, P. Development of a Chemically Defined Medium for in vitro Expansion of Primary Bovine Satellite Cells. Front. Bioeng. Biotechnol. ;Volume 10–2022. (2022). Skrivergaard, S. et al. A simple and robust serum-free media for the proliferation of muscle cells. Food Res. Int. 172 , 113194 (2023). Judson, R. N. & Rossi, F. M. V. Towards stem cell therapies for skeletal muscle repair. npj Regenerative Med. 5 (1), 10 (2020). Buchanan, S. M. et al. Pro-myogenic small molecules revealed by a chemical screen on primary muscle stem cells. Skelet. Muscle . 10 (1), 28 (2020). Relaix, F. et al. Perspectives on skeletal muscle stem cells. Nat. Commun. 12 (1), 692 (2021). Pang, K. T. et al. Insight into muscle stem cell regeneration and mechanobiology. Stem Cell Res. Ther. 14 (1), 129 (2023). Liu, S. et al. Species variations in muscle stem cell-mediated immunosuppression on T cells. Sci. Rep. 14 (1), 23410 (2024). Chen, H., Jia, Y. & Guo, Q. Chapter 6 - Polysaccharides and polysaccharide complexes as potential sources of antidiabetic compounds: A review. In: (ed Atta ur, R.) Studies in Natural Products Chemistry. 67: Elsevier; 199–220. (2020). Mahmoud, Y. A. G., El-Naggar, M. E., Abdel-Megeed, A. & El-Newehy, M. Recent Advancements in Microbial Polysaccharides: Synthesis and Applications. Polymers-Basel 13 , 23 (2021). Zhao, J. et al. Polysaccharide conjugate vaccine: A kind of vaccine with great development potential. Chin. Chem. Lett. 32 (4), 1331–1340 (2021). Shen, J. et al. Effect of Angelica polysaccharide on mouse myeloid-derived suppressor cells. Front. Immunol. 13 , 989230 (2022). Ye, R. et al. Eucommia ulmoides polysaccharide modified nano-selenium effectively alleviated DSS-induced colitis through enhancing intestinal mucosal barrier function and antioxidant capacity. J. Nanobiotechnol. 21 (1), 222 (2023). Yuan, D., Li, C., Huang, Q., Fu, X. & Dong, H. Current advances in the anti-inflammatory effects and mechanisms of natural polysaccharides. Crit. Rev. Food Sci. Nutr. 63 (22), 5890–5910 (2023). Lee, J-H. et al. Effect of Crude Polysaccharides from Ecklonia cava Hydrolysate on Cell Proliferation and Differentiation of Hanwoo Muscle Stem Cells for Cultured Meat Production. Foods 13 (4), 563 (2024). Shin, T. Y., Lee, M. R., Kim, J. C., Nai, Y. S. & Kim, J. S. A new strategy using entomopathogenic fungi for the control of tree borer insects. Entomol. Res. 52 (7), 327–333 (2022). Ng, T. B. & Wang, H. X. Pharmacological actions of Cordyceps, a prized folk medicine. J. Pharm. Pharmacol. 57 (12), 1509–1519 (2005). Tuli, H. S., Sandhu, S. S. & Sharma, A. K. Pharmacological and therapeutic potential of Cordyceps with special reference to Cordycepin. 3 Biotech. 4 (1), 1–12 (2014). Mantzoukas, S. & Eliopoulos, P. A. Endophytic Entomopathogenic Fungi: A Valuable Biological Control Tool against Plant Pests. Appl. Sci-Basel ; 10 (1). (2020). Wu, S. Y. et al. Destruxin B Suppresses Drug-Resistant Colon Tumorigenesis and Stemness Is Associated with the Upregulation of miR-214 and Downregulation of mTOR/beta-Catenin Pathway. Cancers (Basel) ; 10 (10). (2018). Wu, N. et al. A review on polysaccharide biosynthesis in Cordyceps militaris. Int. J. Biol. Macromol. 260 (Pt 1), 129336 (2024). Chen, L. S. et al. A polysaccharide from mycelia of Structural characterization, inhibition on α-glucosidase and improvement of insulin resistance in HepG2 cells. Process. Biochem. 125 , 212–221 (2023). Park, Y., Choi, S., Kim, B. & Lee, S. G. Effects of Extracts on Macrophage as Immune Conductors. Appl. Sci-Basel ; 11 (5). (2021). Yoon, S. Y., Lindroth, A. M., Kwon, S., Park, S. J. & Park, Y. J. Adenosine derivatives from Cordyceps exert antitumor effects against ovarian cancer cells through ENT1-mediated transport, induction of AMPK signaling, and consequent autophagic cell death. Biomed. Pharmacother . 153 , 113491 (2022). Choi, S., Ferrari, G. & Tedesco, F. S. Cellular dynamics of myogenic cell migration: molecular mechanisms and implications for skeletal muscle cell therapies. EMBO Mol. Med. 12 (12), e12357 (2020). Gonzalez, M. L., Busse, N. I., Waits, C. M. & Johnson, S. E. Satellite cells and their regulation in livestock. J. Anim. Sci. 98 (5), skaa081 (2020). Zygmunt, K., Otwinowska-Mindur, A., Piórkowska, K. & Witarski, W. Influence of media composition on the level of bovine satellite cell proliferation. Animals 13 (11), 1855 (2023). Matson, J. P. & Cook, J. G. Cell cycle proliferation decisions: the impact of single cell analyses. FEBS J. 284 (3), 362–375 (2017). Qu, F., Guilak, F. & Mauck, R. L. Cell migration: implications for repair and regeneration in joint disease. Nat. Rev. Rheumatol. 15 (3), 167–179 (2019). Ding, S. et al. Maintaining bovine satellite cells stemness through p38 pathway. Sci. Rep. 8 (1), 10808 (2018). Rastedt, W., Blumrich, E. M. & Dringen, R. Metabolism of Mannose in Cultured Primary Rat Neurons. Neurochem Res. 42 (8), 2282–2293 (2017). Wang, H. H. et al. Nobiletin Prevents D-Galactose-Induced C2C12 Cell Aging by Improving Mitochondrial Function. Int. J. Mol. Sci. 23 , 19 (2022). Jang, J. W. Antiviral activity of polysaccharide derived from entomopathogenic fungus (Jeonbuk National University, 2025). Kim, J. K., Cho, M. L., Karnjanapratum, S., Shin, I. S. & You, S. G. In vitro and in vivo immunomodulatory activity of sulfated polysaccharides from Enteromorpha prolifera. Int. J. Biol. Macromol. 49 (5), 1051–1058 (2011). Kim, H. S. et al. Activation of macrophages by polysaccharide isolated from Paecilomyces cicadae through toll-like receptor 4. Food Chem. Toxicol. 50 (9), 3190–3197 (2012). Huang, L., Shen, M., Morris, G. A. & Xie, J. Sulfated polysaccharides: Immunomodulation and signaling mechanisms. Trends Food Sci. Technol. 92 , 1–11 (2019). Lee, D. Y. et al. The roles of media ingredients in muscle cell culture for cultured meat production-A mini-review. Future Foods ; 10 . (2024). Wang, Y., Zou, L., Liu, W. & Chen, X. An overview of recent progress in engineering three-dimensional scaffolds for cultured meat production. Foods 12 (13), 2614 (2023). He, H. et al. PDLIM5 affects chicken skeletal muscle satellite cell proliferation and differentiation via the p38-MAPK pathway. Animals 11 (4), 1016 (2021). Li, X. et al. Effect of IGF1 on Myogenic Proliferation and Differentiation of Bovine Skeletal Muscle Satellite Cells Through PI3K/AKT Signaling Pathway. Genes 15 (12), 1494 (2024). Long, X. Y., Yan, Q., Cai, L. J., Li, G. Y. & Luo, X. G. Box-Behnken design-based optimization for deproteinization of crude polysaccharides in berry residue using the Sevag method. Heliyon ; 6 (5). (2020). Han, J. H., Yu, J. S., Kim, D. H. & Choi, H. W. The characteristics of bovine satellite cells with highly scored genomic estimated breeding value. J. Anim. Reprod. Biotechnol. 38 (3), 177–187 (2023). Han, J. H. et al. Comparative Analysis of Different Extracellular Matrices for the Maintenance of Bovine Satellite Cells. Animals 14 (23), 3496 (2024). Noë, S. et al. The Myotube Analyzer: how to assess myogenic features in muscle stem cells. Skelet. Muscle . 12 (1), 12 (2022). Additional Declarations No competing interests reported. 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12:49:07","extension":"jpeg","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":301613,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7935981/v1/115eb8933318889cd76ae25a.jpeg"},{"id":96247827,"identity":"f0bec669-07e0-4ea8-877f-1aedd2fcfcb0","added_by":"auto","created_at":"2025-11-19 07:27:44","extension":"jpeg","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3690,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7935981/v1/8b60d2392e82595db2320ddb.jpeg"},{"id":96248042,"identity":"de7e3b86-a735-4470-b18d-1364c6cb714d","added_by":"auto","created_at":"2025-11-19 07:27:58","extension":"jpeg","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1074,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage11.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7935981/v1/696d8e77e56a4d21884c8a7a.jpeg"},{"id":96087572,"identity":"6ff8c90c-8f01-4b04-9a4c-a5b36d5abfa9","added_by":"auto","created_at":"2025-11-17 12:49:07","extension":"jpeg","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":789812,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7935981/v1/2b34c62ad4a27cd6c97a499a.jpeg"},{"id":96248969,"identity":"1b15323a-32e3-4418-b319-d27e38668b16","added_by":"auto","created_at":"2025-11-19 07:29:49","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":15148,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7935981/v1/5be234e3fc2c6e7697d34f76.png"},{"id":96087558,"identity":"362dc046-8624-4de8-baf6-bffb0e12b269","added_by":"auto","created_at":"2025-11-17 12:49:07","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1468,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-7935981/v1/22e931a3ed73b4eac090ac44.png"},{"id":96248562,"identity":"68f63940-4f3c-48d8-a214-d0f33606b89c","added_by":"auto","created_at":"2025-11-19 07:28:40","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":935,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-7935981/v1/7407932a823a24d861ca2709.png"},{"id":96087565,"identity":"0c2787c5-06d7-4470-9b7c-436079c0dad2","added_by":"auto","created_at":"2025-11-17 12:49:07","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":162259,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-7935981/v1/4fe5b52dbaeb96deaf39643b.png"},{"id":96249934,"identity":"113977ea-a7b6-4362-8a04-13c2900d03ed","added_by":"auto","created_at":"2025-11-19 07:36:48","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":935,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-7935981/v1/c89f67573056c473960f6bef.png"},{"id":96087561,"identity":"25157e17-cb1d-4b0f-9d79-2a1b9eb74c0d","added_by":"auto","created_at":"2025-11-17 12:49:07","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":409566,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7935981/v1/2521a2ec95d31e0115498ae8.png"},{"id":96087570,"identity":"720d118c-34f1-4f17-81e7-8768523ee1b1","added_by":"auto","created_at":"2025-11-17 12:49:07","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1456,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7935981/v1/0e129872862c49633d0fb2e5.png"},{"id":96249736,"identity":"db08e470-cd1c-4b75-afe8-03b757636764","added_by":"auto","created_at":"2025-11-19 07:36:10","extension":"png","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":935,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-7935981/v1/6387443998b3e7ba366918ff.png"},{"id":96087562,"identity":"70a048c3-ee53-42e0-945e-bb65d77df287","added_by":"auto","created_at":"2025-11-17 12:49:07","extension":"png","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":68664,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7935981/v1/3aa8b391ac252833935d47c5.png"},{"id":96250097,"identity":"6529aaaa-7556-4f4a-82eb-5e1a881b1188","added_by":"auto","created_at":"2025-11-19 07:37:26","extension":"png","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1521,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7935981/v1/30532fa5d073c7bb7506e84d.png"},{"id":96249686,"identity":"6523a4fd-99ee-4643-bdb7-a218fd1890bb","added_by":"auto","created_at":"2025-11-19 07:36:00","extension":"png","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":935,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-7935981/v1/8f576178b426df65ea658e64.png"},{"id":96087571,"identity":"b31d376d-e4fb-49e5-80af-8fb23b8759e8","added_by":"auto","created_at":"2025-11-17 12:49:07","extension":"png","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":157076,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7935981/v1/988859add68e3e0b1317c8ab.png"},{"id":96087574,"identity":"ea6ab408-1980-413a-8342-27ccb6efd187","added_by":"auto","created_at":"2025-11-17 12:49:07","extension":"xml","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":130724,"visible":true,"origin":"","legend":"","description":"","filename":"a876ef6e129c462e9a046bb92d056e891structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7935981/v1/f221b3208774a88121f559ad.xml"},{"id":96087573,"identity":"972bb88e-a8bb-44fc-bd51-ed760f0ae05d","added_by":"auto","created_at":"2025-11-17 12:49:07","extension":"html","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":144222,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7935981/v1/e4280ea9996e3487254ff631.html"},{"id":96087538,"identity":"2b528244-85cb-487f-9c39-587c3747f0d0","added_by":"auto","created_at":"2025-11-17 12:49:06","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":37411,"visible":true,"origin":"","legend":"\u003cp\u003eScreening of microbial polysaccharides concentration on the viability of bovine satellite cells (BSCs)\u003c/p\u003e\n\u003cp\u003e(A) Cell proliferation of BSCs treated with various concentrations of MP15R polysaccharide, measured using the CCK-8 assay (p \u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003e(B) Cell proliferation following treatment with polysaccharide \u003cem\u003eTolypocladium cylindrosporum\u003c/em\u003e 15 (T15) (p \u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003e(C) Cell proliferation following treatment with polysaccharide \u003cem\u003eTolypocladium cylindrosporum\u003c/em\u003e 237 (T237) (p \u0026lt; 0.001). All values are presented as means ± SE (n = 3). Different letters (a–f) indicate statistically significant differences among groups based on Duncan’s multiple range test. This screening step was used to identify the most effective polysaccharide for subsequent analyses.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7935981/v1/0f86e6f635c1a19807136341.jpg"},{"id":96087539,"identity":"ce79b9e4-6d06-4afb-8d38-1b22b7c13b1e","added_by":"auto","created_at":"2025-11-17 12:49:06","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":112106,"visible":true,"origin":"","legend":"\u003cp\u003eLong-term proliferation and migration of bovine satellite cells (BSCs) treated with Metarhizium pinghaense 15R (MP15R)\u003c/p\u003e\n\u003cp\u003e(A) Morphological images of BSCs at passages 3, 5, 7, and 9 under control and MP15R -treated conditions.\u003c/p\u003e\n\u003cp\u003e(B) Cell proliferation based on direct cell counting. A statistically significant difference was observed only at passage 7 (*p \u0026lt; 0.05). Values are presented as the means ± SE (n = 3).\u003c/p\u003e\n\u003cp\u003e(C) Wound healing assay images captured at 0, 12, and 24 h. Blue lines indicate the wound boundaries.\u003c/p\u003e\n\u003cp\u003e(D) Cell migration index (%) calculated using ImageJ. A significant difference between groups was observed at 24 h (**p \u0026lt; 0.01). Values are presented as the means ± SE (n = 4)\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7935981/v1/9799de6e3a8f5b3c206d7546.jpg"},{"id":96087541,"identity":"b5009c4e-c0f3-4ae9-8c88-c3fd07fb9448","added_by":"auto","created_at":"2025-11-17 12:49:07","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":52889,"visible":true,"origin":"","legend":"\u003cp\u003eCell cycle analysis of bovine satellite cells (BSCs) treated with Metarhizium pinghaense 15R (MP15R) or negative control (NC)\u003c/p\u003e\n\u003cp\u003e(A) Representative flow cytometry plots showing the cell cycle profiles of control and MP15R -treated BSCs.\u003c/p\u003e\n\u003cp\u003e(B) Quantification of the percentage of cells in G0/G1, S, and G2/M phases. Significant differences between groups were observed in G0/G1 (p3, *p \u0026lt; 0.05), S (p3, ***p \u0026lt; 0.001; p5, *p \u0026lt; 0.05), and G2/M (p3 and p5, **p \u0026lt; 0.01). Values are presented as the means ± SE (n = 3).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7935981/v1/79a0cd412e65a9a5024d5655.jpg"},{"id":96087540,"identity":"54af5b07-5e55-4d01-837e-8cc78b094115","added_by":"auto","created_at":"2025-11-17 12:49:06","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":83690,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of PAX7 and MYOD1 in long-term cultured BSCs treated with Metarhizium pinghaense 15R (MP15R)\u003c/p\u003e\n\u003cp\u003e(A) Immunocytochemical staining of PAX7 and MYOD1 in BSCs cultured with or without MP15R at early passage.\u003c/p\u003e\n\u003cp\u003e(B) Quantification of PAX7⁺ cells was performed based on double immunostaining with PAX7 and MYOD1. The number of PAX7-positive nuclei was counted and expressed as a percentage of total DAPI-stained nuclei (*p \u0026lt; 0.05)\u003c/p\u003e\n\u003cp\u003e(C) mRNA expression levels of PAX7 and MYOD1 at passages 3, 5, 7, and 9 as measured by qPCR. Expression levels were normalized to that of GAPDH and presented relative to that of the control group (PAX7, p3, *p \u0026lt; 0.05; MYOD1, p3, **p \u0026lt; 0.01, p7, ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7935981/v1/040b08d4b0d3f43457f09a63.jpg"},{"id":96246756,"identity":"56612d7f-6afb-4701-add2-7dda45ddd5ab","added_by":"auto","created_at":"2025-11-19 07:26:38","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":76830,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Metarhizium pinghaense 15R (MP15R) on the differentiation of bovine satellite cells (BSCs)\u003c/p\u003e\n\u003cp\u003e(A) Immunocytochemistry of MYHC (green, GFP) and nuclei (blue, DAPI) after 4 days of differentiation. Cells were subjected to five conditions: (1) Growth Medium (GM) for 2 days → Differentiation Medium (DM) for 2 days (GM+DM), (2) GM 2 days → DM with 1 µg/mL MP15R for 2 days (GM+1 µg), (3) GM → DM + 3 µg/mL (GM+3 µg), (4) GM → DM + 5 µg/mL (GM+5 µg), and (5) GM + 5 µg/mL for 2 days → DM for 2 days (5 µg+DM). Representative images are shown in the order of MYHC (GFP), DAPI, and merged signals.\u003c/p\u003e\n\u003cp\u003e(B) Fusion index calculated by quantifying the number of DAPI-stained nuclei within MYHC-positive myotubes based on ICC images.\u003c/p\u003e\n\u003cp\u003e(C) mRNA expression levels of MYHC and MYOG were measured by qPCR under the same five conditions. Values are presented as means ± SE (n = 3). Different letters (a–c) indicate statistically significant differences among groups according to Duncan’s multiple range test (Fusion Index, p \u0026lt; 0.05; MYHC, p \u0026lt; 0.05; MYOG, p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7935981/v1/5d034f33579275bb79b9e6d1.jpg"},{"id":96363736,"identity":"3b61bbdd-7c11-4411-bc9c-9ca87614b7a4","added_by":"auto","created_at":"2025-11-20 10:07:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1312346,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7935981/v1/96c87de5-64e0-485d-b446-c5618ced720a.pdf"},{"id":96249765,"identity":"8a69ecf0-7080-4bc8-bb78-5fe29c53f68e","added_by":"auto","created_at":"2025-11-19 07:36:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":103811,"visible":true,"origin":"","legend":"","description":"","filename":"ParkSupplementaryS1ScientificReports2025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7935981/v1/8b4fb202cf14f945dd98d8e1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Polysaccharides from Metarhizium pinghaense: a novel biomaterial to modulate bovine satellite cell fate for cultured meat","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eWith the rise in global meat consumption, concern over the environmental impact and sustainability of traditional livestock farming is increasing. Therefore, cultured meat has emerged as a promising alternative that offers potential benefits in terms of resource efficiency, animal welfare, and greenhouse gas reduction.(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) Efficient large-scale production of muscle tissue is essential in the cultured meat industry because muscle is not only the primary structural and nutritional component of meat but also a key factor in reducing the overall manufacturing costs and meeting consumer expectations.(\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) As a protein-dense and nutritionally vital component, muscle is central to the nutritional value of cultured meat, and its mass production is essential for meeting both the nutritional requirements of the growing population and consumer expectations.(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eSatellite cells are muscle stem cells located between the basal lamina and sarcolemma, and they play a key role in muscle regeneration and maintenance. These cells remain in a quiescent state and become activated in response to muscle injury or other physiological signals.(\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) Upon activation, satellite cells proliferate and differentiate to form new muscle fibers or fuse with existing fibers to maintain and repair muscle tissue.(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) Satellite cell functions are regulated by transcription factors, such as paired box 7 (PAX7), and their proliferation and differentiation are significantly influenced by signaling pathways, such as Notch and Wnt.(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) In addition, satellite cell activation and differentiation are modulated by various external cues, including inflammatory responses, growth factors, and mechanical stimuli. Dysregulation of these processes can lead to muscle atrophy and muscle-related diseases.(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e) Muscle stem cells proliferate for a certain period of time and then stop proliferating, or their function deteriorates with cell aging.(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) Appropriate culture medium and nutrients are required to promote the proliferation of muscle stem cells. However, a clear mechanism for cell proliferation has not yet been fully elucidated, and continuous improvements in culture conditions are needed.(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eRecent studies have attempted to overcome the challenges of muscle stem cell proliferation by treatment with small molecules or optimization of culture media composition. For instance, the inhibition of p38 mitogen-activated protein kinase using small molecules such as SB203580 has been shown to enhance the self-renewal capacity of muscle stem cells and maintain their regenerative potential during ex vivo expansion.(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e) Additionally, receptor tyrosine kinase inhibitors, such as CEP-701, can promote satellite cell expansion and improve muscle regeneration.(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e) However, the large-scale production of muscle stem cells remains a significant challenge. Traditional culture methods often lead to the spontaneous differentiation or senescence of muscle stem cells, limiting their proliferative capacity and therapeutic potential.(\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e)\u003c/p\u003e\u003cp\u003ePolysaccharides have attracted attention as potential alternatives for overcoming these limitations owing to their broad spectrum of biological activities. They are naturally occurring polymers found in a wide range of organisms, including plants, animals, algae, fungi, and bacteria.(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) In particular, microbially derived polysaccharides can be produced in large quantities within a short time period, making them highly cost-effective and a subject of active research.(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) Moreover, because of their diverse physiological activities, natural polysaccharides have the potential to improve cellular functions such as alleviating colitis and upregulating immune cells derived from mouse bone marrow.(\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) For example, crude polysaccharides extracted from Ecklonia cava have been reported to enhance the proliferation and differentiation of Hanwoo muscle satellite cells by activating pathways such as hepatocyte growth factor, its receptor MET, and focal adhesion kinase.(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eEntomopathogenic fungi are a group of microorganisms that infect and ultimately kill insects by attaching to the insect cuticle, penetrating the host body, and causing death within a few days.(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) Among the approximately 80,000 known fungal species, only approximately 700 are entomopathogenic and exhibit a unique life cycle that involves parasitizing and killing insect hosts. These fungi are considered harmless to humans and characterized by high environmental stability and host specificity, making them promising candidates as biological control agents to replace conventional chemical pesticides.(\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) Some entomopathogenic fungi belonging to the family Cordycipitacege, including Cordyceps, form fruiting bodies after killing their insect hosts. These Cordyceps possess high pharmacological value and exhibit anti-inflammatory, antioxidant, hypoglycemic, and immunomodulatory activities; thus, have been widely used in traditional medicine.(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e) Owing to these beneficial properties, entomopathogenic fungi and Cordyceps have been actively studied and developed as pharmaceutical and nutraceutical agents.(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e) In addition to their secondary metabolites, such as cordycepin and destruxin,(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) which are known for their pharmacological effects, entomopathogenic fungi also produce large amounts of bioactive polysaccharides with significant therapeutic potential. Polysaccharides derived from Metarhizium taii have been reported to induce insulin resistance in HepG2 cells,(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) whereas those from Cordyceps militaris exhibit antitumor and immune-enhancing effects.(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e) Although these findings indicate the biological and pharmacological potential of entomopathogenic fungal polysaccharides, their applicability in cell culture systems remains poorly understood. In particular, the use of these polysaccharides in muscle stem cells has not yet been thoroughly investigated. Although preliminary studies suggest potential benefits, comprehensive research is required to elucidate the mechanisms underlying the influence of polysaccharides on muscle stem cell behavior and assess their efficacy in large-scale cell culture systems.\u003c/p\u003e\u003cp\u003eIn the present study, we investigated the effects of polysaccharides derived from entomopathogenic fungi on the proliferation and differentiation of bovine satellite cells (BSCs). For this purpose, we employed two fungal strains from Tolypocladium cylindrosporum (T15 and T237) and one strain from Metarhizium pinghaense 15R (MP15R), which were previously observed to produce high levels of polysaccharides. The results demonstrated that the polysaccharides derived from these entomopathogenic fungi significantly enhanced BSCs proliferation and promoted the expression of muscle differentiation markers, such as myosin heavy chain (MYHC) and myogenin (MYOG), thereby facilitating the proliferation and differentiation of BSCs. These findings suggest novel strategies for harnessing their potential for cultured meat production and regenerative medicine.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eScreening of microbial polysaccharides identifies MP15R as a potent enhancer of BSCs viability\u003c/h2\u003e\u003cp\u003eTo assess the cytotoxicity of the polysaccharides, the BSCs were treated with the polysaccharides at various concentrations (0.05\u0026ndash;20 \u0026micro;g/mL), and then CCK-8 assays were performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). All experiments were performed in triplicate (n\u0026thinsp;=\u0026thinsp;3) and statistical analyses were performed using Duncan\u0026rsquo;s multiple comparison test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The results revealed that cell viability in the 0.05 \u0026micro;g/mL, 10 \u0026micro;g/mL, 5 \u0026micro;g/mL, and 20 \u0026micro;g/mL MP15R polysaccharide groups was higher than that of the NC. The highest cell viability was observed at a concentration of 5 \u0026micro;g/mL, which was approximately 23.8% higher than that of the NC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In the polysaccharide T15 treatment group, the highest cell viability was observed at 500 ng/mL, which was similar to that of the NC group. However, when treated with a high concentration of 500 ng/mL or higher, cell viability tended to gradually decrease (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Finally, in the polysaccharide T237 treatment group, the highest cell viability was observed at a concentration of 100 ng/mL, although the cell viability was approximately 10% lower than that of the NC group. When treated with a high concentration of 100 ng/mL or higher, the cell viability tended to gradually decrease (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Based on these results, MP15R polysaccharide at 5 \u0026micro;g/mL was selected for subsequent experiments due to its superior effect on BSC proliferation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMP15R polysaccharide promotes long-term proliferation and enhances the migratory capacity of BSCs\u003c/h3\u003e\n\u003cp\u003eBased on the previous screening results, MP15R polysaccharide at a concentration of 5 \u0026micro;g/mL was identified as the most effective condition for promoting BSC proliferation and thus was selected for further evaluation of its long-term effects. First, to assess these effects, the morphological characteristics and cell proliferation were evaluated at passages 3, 5, 7, and 9. Morphological evaluation showed no obvious visual differences between the NC and MP15R polysaccharide-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Under both conditions, the cells exhibited a typical spindle-shaped morphology, and the cell density gradually decreased with passage. Cell proliferation, as assessed by the cell count, was consistently higher in the treated group than in the NC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This difference was statistically significant at passage 7 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), suggesting a potential growth-promoting effect of the MP15R polysaccharide at this stage. Second, since cell migration plays a key role in biological processes such as muscle differentiation, development, and tissue repair, a wound-healing assay was conducted to evaluate the effect of the MP15R polysaccharide on the migration of BSCs.(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e) The assay was performed at passage 3 to determine whether the additive influenced the migratory capacity of the cells at an early stage. Morphologically, the wounds appeared narrower in the MP15R polysaccharide-treated group (5 \u0026micro;g/mL) compared to the NC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). To quantify this, the wound area was measured using ImageJ (version 1.54g, National Institutes of Health, USA). A comparison of scratch distances between 0 and 24 h revealed a migration index of 49.26% in the NC group and 60.13% in the MP15R polysaccharide-treated group, with the difference being statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). This enhanced migration suggests a possible regulatory role of the MP15R polysaccharide in the proliferation and differentiation of BSCs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eMP15R treatment accelerates cell cycle progression by promoting S-phase entry\u003c/h3\u003e\n\u003cp\u003eAnalysis of the cell cycle phase distribution of the BSC population is a direct method of measuring how many cells are activated and how many are dormant.(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e) To assess the impact of the MP15R polysaccharide on the proliferative capacity of BSCs, a cell cycle analysis was conducted. Previous studies have demonstrated that alterations in the extracellular matrix or signaling pathways can influence the distribution of BSCs across different cell cycle phases, thereby affecting their proliferation and differentiation potential. The cell cycle analysis was performed at passages 3 and 5. In the G0/G1 phase, both p3 and p5 were slightly higher in the treatment group (5 \u0026micro;g/mL) than in the NC group, with a significant difference between the p3 NC groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The S phase was 4.3% and 3.9% in the p3 and p5 NC groups, while it was 7.3% and 5.6% in the p3 and p5 treatment groups (5 \u0026micro;g/mL). The S phase gradually decreased with the passage, but the population of the treatment group (5 \u0026micro;g/mL) was significantly higher than that of the NC group (p3, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; p5, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The G2/M phase naturally decreased as the G0/G1 and S phases increased, suggesting that the overall proliferation activity was enhanced by rapid cycle rotation (p3, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; p5, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Our results indicate that treatment with the MP15R polysaccharide can influence the proliferation and cell cycle dynamics of BSCs during passages 3 and 5, suggesting its potential impact on both short- and long-term cellular behavior.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eMP15R polysaccharide preserves the undifferentiated state of BSCs during extended culture\u003c/h3\u003e\n\u003cp\u003eThe expression patterns of PAX7 and MYOD1 in BSCs were examined using immunocytochemistry (ICC) and quantitative PCR (qPCR). ICC showed positive staining for both markers in NC and MP15R polysaccharide-treated groups at passage. Although both proteins were visualized by double immunostaining, only PAX7-positive nuclei were quantitatively analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The proportion of PAX7\u0026thinsp;+\u0026thinsp;cells, expressed as a percentage of the total DAPI-stained nuclei, was higher in the MP15R polysaccharide-treated group than in the NC group, indicating that the MP15R polysaccharide may support the preservation of stemness in satellite cells through the early upregulation of PAX7 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). qPCR analysis revealed that PAX7 mRNA levels remained consistently higher in the MP15R polysaccharide-treated group throughout passages 3, 5, 7, and 9, with a significant increase observed at passage 3 (PAX7, p3, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; MYOD1, p3, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In contrast, MYOD1 expression displayed a more variable pattern, with significantly higher expression in the MP15R polysaccharide-treated group and NC group at passages 3 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and 7 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). No significant differences were observed between passages 5 and 9. These findings indicate that MP15R polysaccharides may enhance early myogenic activation, particularly by upregulating PAX7 and MYOD1 at the early stages, while exerting a more limited or passage-dependent effect on MYOD1 expression.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eMP15R polysaccharide enhances the terminal myogenic differentiation of BSCs\u003c/h3\u003e\n\u003cp\u003eThe effect of MP15R polysaccharide on BSC differentiation was assessed by adding the polysaccharide to the differentiation medium at various concentrations. Since 5 \u0026micro;g/mL MP15R polysaccharide showed a positive effect on proliferation in the growth medium, an additional experiment was conducted in which cells were first cultured in growth medium containing 5 \u0026micro;g/mL MP15R polysaccharide and then switched to differentiation medium. After four days of differentiation under the five different treatment conditions, MYHC expression and myotube formation were assessed by ICC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). MYHC-positive multinucleated myotubes were observed in all groups. However, no obvious morphological differences in myotube formation were observed among the groups, including the NC and MP15R polysaccharide-treated groups. Quantification of the fusion index, calculated as the percentage of DAPI-stained nuclei within MYHC-positive myotubes, revealed statistically significant increases in the GM\u0026thinsp;+\u0026thinsp;1 \u0026micro;g and 5 \u0026micro;g\u0026thinsp;+\u0026thinsp;DM groups compared to the NC (GM\u0026thinsp;+\u0026thinsp;DM) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). To examine the effect of the MP15R polysaccharide on gene expression, the mRNA levels of MYHC and MYOG were analyzed by qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Both genes were significantly upregulated in the GM\u0026thinsp;+\u0026thinsp;1 \u0026micro;g and 5 \u0026micro;g\u0026thinsp;+\u0026thinsp;DM groups relative to the NC. Statistical analysis using Duncan\u0026rsquo;s multiple range test identified distinct groupings (a-c), thus confirming significant differences in gene expression among the treatment conditions (MYHC, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; MYOG, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). These results suggest that the MP15R polysaccharide promotes BSC differentiation at the molecular level, particularly when applied during early differentiation or following pretreatment in a growth medium.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eAs the demand for scalable and sustainable cultured meat production grows, increasing attention has been focused on biomaterials capable of supporting muscle stem cell expansion.(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) Natural polysaccharides, known for their bioactivity in mammalian systems, such as immune modulation and tissue regeneration, have been used as supplements in various cell culture contexts.(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e) However, their specific effects on BSCs remain poorly characterized, and to our knowledge, no previous study has investigated the dual role of fungal-derived polysaccharides in promoting both proliferation and differentiation of BSCs under long-term culture conditions.\u003c/p\u003e\u003cp\u003eIn this study, we examined the effects of a fungal polysaccharide derived from strain MP15R on the proliferation, migration, cell cycle, and differentiation of BSCs. Our results demonstrated that the MP15R polysaccharide significantly improved cell viability in a dose-dependent manner, with 5 \u0026micro;g/mL identified as the optimal concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). This suggests that treatment with an optimal concentration of the polysaccharide exerts beneficial effects on cell culture. In long-term cultures, BSCs treated with the MP15R polysaccharide exhibited consistently higher cell numbers than those in the NC group, with a statistically significant difference at passage 7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Thus, the MP15R polysaccharide may support prolonged expansion without early senescence.\u003c/p\u003e\u003cp\u003eIncreased S-phase populations observed via flow cytometric analysis further confirmed the proliferative effect of the MP15R polysaccharide, particularly in early passages. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, a significant increase in the S-phase population was observed at passages 3 and 5 in the MP15R polysaccharide-treated group, suggesting enhanced entry into the cell cycle and elevated DNA synthesis. Notably, the decrease in G2/M phase may have reflected accelerated cell cycle progression, resulting in increased proliferation. Although the S-phase proportion decreased at passage 5 compared to passage 3, this may indicate a passage-dependent decline in responsiveness to the MP15R polysaccharide, potentially due to differential utilization across specific cell cycle phases.(\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e) Additionally, BSCs cultured with the MP15R polysaccharide demonstrated significantly enhanced migration capacity, as evidenced by a higher migration index in wound-healing assays at 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Enhanced motility may contribute to faster tissue formation and improved responsiveness to regenerative cues in vitro.(\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eGene and protein expression analyses revealed that MP15R polysaccharide treatment increased PAX7 expression in both early and late passages, and ICC confirmed a higher population of PAX7\u0026thinsp;+\u0026thinsp;nuclei (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). PAX7 is a key transcription factor that maintains the identity and self-renewal ability of BSCs. Its sustained expression suggests that the MP15R polysaccharide preserves the stemness of BSCs during long-term expansion.(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e) While MYOD1 expression was transiently elevated at early passages (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), it declined by passage 7 in the treated cells, suggesting a controlled or delayed commitment to differentiation.\u003c/p\u003e\u003cp\u003eInterestingly, the fusion index and expression of myogenic differentiation markers (MYHC and MYOG) were significantly upregulated when the MP15R polysaccharide was administered at specific stages during the differentiation protocol. In particular, the 5 \u0026micro;g\u0026thinsp;+\u0026thinsp;DM group (pretreatment in growth media followed by normal differentiation) showed significant increases in both the fusion index and gene expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These results suggest that the MP15R polysaccharide supports not only the proliferation but also the differentiation potential of BSCs when applied at the appropriate time points.\u003c/p\u003e\u003cp\u003eCompositional analysis of MP15R polysaccharide revealed that its predominant monosaccharides are galactose (52.11 mg/g), mannose (48.53 mg/g), arabinose (4.16 mg/g), and fucose (2.86 mg/g), while glucose was not detected. Mannose is efficiently metabolized by cultured cerebellar granule neurons,(\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e) while D-galactose induces cellular senescence in C2C12 myoblasts.(\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e) However, the specific effects of galactose and mannose on muscle stem cells remain largely unexplored, making it difficult to predict how the monosaccharide composition of the MP15R polysaccharide influences these cells. In addition, the MP15R polysaccharide is characterized as an anionic polysaccharide containing 18.54% sulfate groups;(\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e) however, the precise mechanisms by which these negatively charged moieties affect muscle stem cells remain unclear. Sulfated polysaccharides are known to possess strong immunomodulatory potential because they regulate macrophages and natural killer (NK) cells and promote homeostasis by enhancing cytokine secretion and immune function.(\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e) Based on these immunological properties, it is plausible that the MP15R polysaccharide exerts beneficial effects on muscle cell growth and immune regulation. Therefore, the observed enhancement in cell proliferation is likely attributable to the combined effects of various saccharide residues and sulfate groups present in the MP15R polysaccharide rather than the action of any single monosaccharide component alone.(\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e) From an applied perspective, this study demonstrated that the MP15R polysaccharide, previously considered a byproduct of the cultivation of entomopathogenic fungi, exhibits meaningful biological activity by promoting cell cycle progression and accelerating the recovery of damaged cells. These findings suggest the potential use of the MP15R polysaccharide as a culture medium supplement to support the growth and differentiation of BSCs. Notably, this polysaccharide holds promise as a novel growth-promoting biomaterial that can overcome the limitations associated with conventional chemical growth factors, such as cost and stability. Furthermore, our findings suggest that it offers a promising strategy for overcoming a major bottleneck in cultured meat bioprocessing by maintaining satellite cell populations that are both expandable and myogenically competent. Traditional culture methods often induce spontaneous differentiation or reduce the regenerative potential of muscle stem cells over time.(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) Our findings indicate that the MP15R polysaccharide may function as a biomaterial additive to address these limitations and improve biomass production from BSCs.\u003c/p\u003e\u003cp\u003eThis study demonstrates that polysaccharides derived from entomopathogenic fungi can significantly enhance the proliferation, migration, and myogenic differentiation of BSCs in vitro. By promoting PAX7 expression and S-phase entry while maintaining stem-like characteristics over multiple passages, the MP15R polysaccharide contributes to the long-term expansion of functional muscle stem cells. In addition, the increased expression of MYHC and MYOG, along with the improved fusion indices under specific treatment conditions, suggests that the MP15R polysaccharide not only preserves the regenerative capacity of BSCs but also primes the cells for efficient differentiation when required (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These findings hold substantial significance for the cultured meat industry, where one of the key challenges lies in maintaining a scalable and renewable source of high-quality muscle cells. The ability of the MP15R polysaccharide to simultaneously support stemness and differentiation under controlled conditions makes it a valuable candidate for developing improved bioprocessing strategies. Furthermore, the use of natural, microbial-derived polysaccharides aligns with the principles of sustainability and cost efficiency, which are critical for the transition from laboratory-scale to industrial-scale cultured meat production.(\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e) Although further studies are required to elucidate the underlying molecular mechanisms and assess their performance in 3D culture systems or bioreactors, our results lay the groundwork for future exploration of polysaccharide-based supplements in muscle stem cell culture. Overall, this study provides novel insights into how functional biomolecules from microbial sources can be repurposed to enhance muscle cell performance and paves the way for innovative approaches in regenerative biology and cell-based meat engineering.\u003c/p\u003e\u003cp\u003eDespite the promising findings, this study has several limitations that should be considered. First, the effects of the MP15R polysaccharide were evaluated exclusively under 2D monolayer conditions. While this approach is suitable for initial screening, it does not fully recapitulate the complex three-dimensional environment of native muscle tissue or the dynamic conditions within an industrial bioreactor. Therefore, future investigations are necessary to validate whether similar benefits are observed in 3D culture systems, such as scaffolds or microcarriers, which would be critical for assessing its practical applicability. Second, while this study characterized the phenotypic outcomes of MP15R treatment, the precise molecular mechanisms underlying its bioactivity were not elucidated. Although the precise molecular mechanisms by which the MP15R polysaccharide acts remain unclear, previous studies have demonstrated that polysaccharides can modulate intracellular signaling pathways, such as mitogen-activated protein kinase and PI3K/AKT, both of which are closely linked to satellite cell proliferation and differentiation.(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e) A comprehensive mechanistic investigation is required to fully understand how MP15R interacts with cellular machinery to regulate cell fate. Clarifying these pathways will be essential for optimizing its application and for the rational design of next-generation biomaterials for cultured meat bioprocessing.\u003c/p\u003e\u003cp\u003eIn conclusion, this study demonstrated that the MP15R polysaccharide promotes BSC proliferation during long-term culture and enhances muscle differentiation efficiency at specific concentrations. Polysaccharides generally have low cytotoxicity and high biocompatibility, which allow them to maintain cell viability and function during long-term culture. These properties suggest their potential as practical supplements for stably supplying the cells required for cultured meat production.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eFungal strain and polysaccharides extraction\u003c/h2\u003e\u003cp\u003eThe entomopathogenic fungus Metarhizium pinghaense 15R (KACC 83065BP) was obtained from the Korean Agricultural Culture Collection (KACC, Korea) and cultured by the Center for Industrialization of Agricultural and Livestock Microorganisms (CIALM, Korea). The dried polysaccharide extract derived from the culture broth was subsequently provided to our laboratory and used for experimental analyses.\u003c/p\u003e\u003cp\u003eTolypocladium cylindrosporum strains T15 and T237 were isolated from sedimentary soils collected from Danyang-gun, Chungcheongbuk-do, and Uljin-gun, Gyeongsangbuk-do, respectively. These strains were stored at \u0026minus;\u0026thinsp;20\u0026deg;C and, for experimental purposes, cultured in Sabouraud dextrose broth (SDB) (238230; BD DIFCO) medium at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C with shaking at 150 rpm for 14 days. The culture broths were centrifuged at 10,000 rpm for 20 min at 4\u0026deg;C to remove the mycelial biomass, followed by filtration through filter paper. The resulting supernatants were collected and polysaccharides were precipitated by adding cold ethanol at three times the volume of the supernatant. The precipitates were recovered by centrifugation under the same conditions, and the residual ethanol was removed.\u003c/p\u003e\u003cp\u003eAll polysaccharides were treated using the Sevag method(\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e) to remove proteins and lipids. The resulting supernatants were further purified using a 0.45 \u0026micro;m syringe filter (SP25P045NL; Hyundai Micro). Cold absolute ethanol (five volumes) was then added, and the solution was incubated at 4\u0026deg;C for 24 h. After ethanol removal, the precipitated polysaccharides were redissolved in distilled water and filtered through a 0.22 \u0026micro;m syringe filter (SP25P020NL; Hyundai Micro).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eEthics Approval\u003c/h2\u003e\u003cp\u003eThis study follows the recommendations of the ARRIVE guidelines. All animal procedures were approved by the Animal Ethics Committee of Jeonbuk National University (JBNU; NON2023-137), Republic of Korea. All experiments were performed in accordance with the ethical guidelines and regulations of the Jeonbuk National University.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eBovine satellite cell isolation\u003c/h2\u003e\u003cp\u003eBovine satellite cells were isolated from the \u003cem\u003elongissimus dorsi\u003c/em\u003e (loin) muscle of a 1-month-old male Korean native calf according to a previously reported protocol (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). Briefly, 5 g of muscle tissue was weighed and finely chopped using surgical scissors for approximately 5 min. The minced tissue was enzymatically digested for 2 h at 37\u0026deg;C in a solution composed of DMEM/F12 (Gibco, Carlsbad, CA, USA, #11320-033), 0.25% trypsin-EDTA (TE) (Gibco, #25200-072), collagenase II (Worthington, Lakewood, NJ, USA, #CLS-2, 5 g), Dispase II (Roche, Indianapolis, IN, USA, #4942078001, 1 U/mL), and 10% antibiotic-antimycotic (A.A., Gibco, #15240062). After digestion, the cell suspension was neutralized using DMEM low glucose (Gibco, #11885092) supplemented with 15% fetal bovine serum (FBS) (Gibco, 16000-044, 26140079) and 1% A.A. and then centrifuged at 80 \u0026times; g for 3 min at 4\u0026deg;C. The supernatant was sequentially filtered through 100 \u0026micro;m and 40 \u0026micro;m strainers. Red blood cells were removed using Red Blood Cell (RBC) lysing buffer (Sigma-Aldrich, St. Louis, MO, USA, #R7757-100mL) and then washed twice with PBS. The remaining cells were resuspended in primary culture media containing Ham\u0026rsquo;s F-10 (Gibco, #11550-043), 20% FBS, 1% A.A., basic fibroblast growth factor (bFGF) (R\u0026amp;D System, Minneapolis, MN, USA, #223-FB-500/CF, 5 ng/mL), and Primocin (Invivogene, Pak Shek Kok, New Territories, Hong Kong, ant-pm-2, 100 \u0026micro;g/mL) until reaching a cell density 70\u0026ndash;80% confluence. Before use, the medium was switched to a formulation without Primocin, and penicillin-streptomycin (PS) was replaced with A.A. The experiment was performed from passages 3 to 9 to evaluate how the polysaccharide treatment influenced the proliferation and maintenance of BSCs over a long-term culture.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eSatellite cell culture and differentiation\u003c/h2\u003e\u003cp\u003ePrimary muscle-derived cells (passage 2) were maintained on culture dishes that had been pre-coated with a 0.1% gelatin solution (G1319; Sigma-Aldrich, St. Louis, MO, USA). For standard expansion, bovine satellite cells (BSCs) were cultured in a growth medium composed of Ham\u0026rsquo;s F-10 (Gibco, #11550-043) supplemented with 20% FBS, 1% Antibiotic-Antimycotic, and 5 ng/mL bFGF. In the experimental groups, the medium was additionally supplemented with the designated polysaccharides during each feeding. The cells were subcultured every four days, with the culture medium being refreshed every two days. To assess long-term proliferation, BSCs were plated onto 6-well plates at a density of 4.8 \u0026times; 10⁴ cells per well. At each subculture from passage 3 (P3) to P9, cells were harvested, and total cell counts for each group were determined in triplicate using a LUNA-FL\u0026trade; Dual Fluorescence Cell Counter (Logos Biosystems, Cat# L20001). The seeding densities for other experimental formats were as follows: 1 \u0026times; 10⁶ cells per 100 mm dish, 1 \u0026times; 10⁴ cells per well for 4-well plates, and 6.4 \u0026times; 10\u0026sup2; cells per well for 96-well plates. To induce myogenic differentiation, BSCs were initially expanded in the growth medium as described. For these experiments, cells were seeded at 9.6 \u0026times; 10⁴ cells per well in 6-well plates or 2 \u0026times; 10⁴ cells per well in 4-well plates. Following an initial 48-hour proliferation period, the culture medium was switched to a differentiation medium (DM), which consisted of DMEM low glucose, 2% Horse Serum, and 1% Antibiotic-Antimycotic. The cells were then maintained in this DM on gelatin-coated dishes for an additional two days to promote differentiation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eCell proliferation analysis\u003c/h2\u003e\u003cp\u003eBovine satellite cells were seeded in a 96-well plate with various concentrations of polysaccharide mixes (MP15R, T15, and T237). The 96-well plate was coated with 0.1% gelatin, and BSCs were seeded in every well at a density of 6.4 \u0026times; 102 cells. The cells were cultured for 3 days, and cell growth was determined using Cell Counting Kit-8 (CCK-8) (#CK04-11; Dojindo, Kumamoto, Japan). Cells were treated with CCK-8 solution according to the manufacturer\u0026rsquo;s instructions and incubated at 37\u0026deg;C for 3 h. Growth was measured using a microplate reader at a wavelength of 450 nm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eGene expression analysis by qRT-PCR\u003c/h2\u003e\u003cp\u003eRNA was extracted from cells using the AccuPrep\u0026reg; Universal RNA Extraction kit (Bioneer, Seoul, Korea) according to the manufacturer\u0026rsquo;s instruction. One microgram of total RNA from each sample was reverse transcribed to cDNA using the Accupower\u0026reg; CycleScript RT Premix (Bioneer, Seoul, Republic of Korea) according to the manufacturer\u0026rsquo;s instruction. Relative gene expression was measured in triplicate using Powerup SYBR Green Master Mix (Applied Biosystems, Waltham, MA, USA). The primer sequences are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. For data normalization, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was employed as the housekeeping gene. Quantitative PCR was performed using a LightCycler\u0026reg; 96 system, and the relative expression levels were determined by the 2⁻ΔCt method.(\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e\u003cp\u003eCultured BSCs were seeded in a 4-well plate at a density of 1 \u0026times; 104 and cultured for four days. After four days, the cells were washed twice with PBS and then fixed with cold 4% paraformaldehyde for 20 min at room temperature. The washing solution was composed of PBS containing 0.3% Triton X-100 (PBST), and the blocking solution was PBST containing 3% bovine serum albumin (BSA; Bovogen, Keilor East, Australia). After fixation, the cells were washed three times with PBS and then incubated in blocking solution for 1 h at room temperature for permeabilization and blocking. After blocking, the cells were washed three times with washing solution. After fixation, cells were then stained overnight at 4\u0026deg;C with the following primary antibodies: anti-MYOD1 (Polyclonal, 1:200, Proteintech, Rosemont, IL, USA), anti-PAX7 (PAX7, monoclonal, 1:50, DHSB, Iowa, IA, USA) and anti-MYHC (MF20, monoclonal, 1:20, Iowa City, IA, USA). Subsequently, cells were then washed three times with washing solution stained with Alexa488 anti-mouse (Invitrogen, USA, A11001) antibodies and Alexa568 labeled anti-rabbit (Invitrogen, USA, A11011) antibodies at room temperature for 2 h and stained with 1 \u0026micro;g/mL of 4\u0026rsquo;,6-diamidino-2-phenylindole (DAPI) for 5 min. After DAPI staining, the cells were washed three times with the washing solution for 10 min. Bovine satellite cells stained with PAX7, MYOD1, and MF20 were captured using a Leica DFC 9000 (Deerfield, IL, USA) at 200x magnification. The expression of PAX7 and MYOD1 was calculated as a ratio of the total DAPI count.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eCell cycle analysis\u003c/h2\u003e\u003cp\u003eBovine satellite cells were collected during passages 3 and 5 and seeded in a 35 mm dish at a density of 4.8 \u0026times; 104. For cell cycle analysis, the cells were detached using 0.25% trypsin-EDTA and neutralized media containing DMEM low glucose, 15% FBS, and 1% A.A. Cells were then washed with cold PBS (containing 1% BSA), fixed with 70% ethanol for 5 min at 4\u0026deg;C, and then stored at \u0026minus;\u0026thinsp;20\u0026deg;C until the day of cell cycle analysis (1\u0026ndash;2 weeks). On the day of cell cycle analysis, cells were centrifuged at 850 \u0026times; g at 4\u0026deg;C for 5 min. The ethanol was removed, and the cells were washed twice with PBS. Afterwards, 100 \u0026micro;g/mL of RNase A (Sigma-Aldrich, #70856) and 25 \u0026micro;g/mL of propidium iodide (PI) (Bio Legend, San Diego, CA, USA, #421301) were added with PBS. The cells were analyzed by flow cytometry using a blue laser (excitation at 488 nm).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003ePAX7\u0026thinsp;+\u0026thinsp;cell population and fusion index percentage measurement\u003c/h2\u003e\u003cp\u003ePAX7\u0026thinsp;+\u0026thinsp;cell populations were quantified by manually counting the number of nuclei costained with DAPI and PAX7 in three randomly selected fields per condition. Percentages were calculated by dividing the number of PAX7\u0026thinsp;+\u0026thinsp;nuclei by the total number of DAPI-stained nuclei in the same field. The cells were seeded in 4-well plates at a density of 1 \u0026times; 104 cells/well. Counting was performed manually using PowerPoint-marked fluorescence images. The fusion index was defined as the percentage of nuclei located within the myotubes, which are defined as multinucleated cells containing three or more nuclei, relative to the total number of nuclei in the same field. Cells were seeded at a density of 2 \u0026times; 104 cells/well and grown in GM for 2 days, followed by the induction of differentiation. Three or more randomly selected fields per condition were analyzed using Myotube Analyzer software (available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/SimonNoe/myotube-analyzer-app).(59)\u003c/span\u003e\u003cspan address=\"https://github.com/SimonNoe/myotube-analyzer-app).(59)\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eCell migration effect (wound-healing assay)\u003c/h2\u003e\u003cp\u003eA wound-healing assay was performed to assess cell migration. BSCs were seeded in 6-well plates at a density of 4.8 \u0026times; 104 cells/well and cultured in growth medium for two days until they reached confluence. A linear scratch was made in each well using a sterile 1000 \u0026micro;L pipette tip, after which the medium was replaced with fresh growth medium to remove detached cells. Images of the scratch areas were captured at 0, 12, and 24 h using a Leica DFC9000 imaging system (Leica Microsystems, Germany) at 50\u0026times; magnification. Cell migration was quantified by measuring wound closure over time, and the migration index was analyzed using the ImageJ software (NIH, Bethesda, MD, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eSTATISTICAL ANALYSIS\u003c/h2\u003e\u003cp\u003eP All experiments were performed three times, and the data from all repetitions of each experiment were collated and expressed as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE) of the mean. Statistical tests were conducted using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA), and statistical differences were analyzed using Student\u0026rsquo;s t-test or analysis of variance (ANOVA) followed by Duncan\u0026rsquo;s multiple range test for post hoc comparisons. Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eDAta availibility\u003c/h2\u003e\u003cp\u003eThe polysaccharides MP15R, T15, and T237 generated in this study are available from the corresponding author (Hyun Woo Choi, [email protected]) with a completed material transfer agreement. Any other data supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003ecode availibility\u003c/h2\u003e\u003cp\u003eThis article does not report original code.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eSeveral authors (Tae-Young Shin, Hyun-Woo Choi, Ji-Hoon Park and Ji-Won Jang) are inventors on a pending Korean patent application (Application No. 10-2025-0069520) related to the use of Metarhizium pinghaense polysaccharides for modulating bovine satellite cell fate described in this manuscript. The patent application is assigned to Jeonbuk National University Industry-Academic Cooperation Foundation, Chungbuk National University Industry-Academic Cooperation Foundation, and the Center for Industrialization of Agricultural and Livestock Microorganisms (CIAM). The other authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003esupplementaRY information\u003c/h2\u003e\u003cp\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, listing primer sequences used for qPCR, is available in the online version of this article.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis research was supported by the \u0026lsquo;High Value-added Food Technology Development Program\u0026rsquo; of the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET), funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA), Republic of Korea (Grant No. 322006-05-CG000).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, T.Y.S. and H.W.C.; methodology, J.H.P., and J.W.J.; Investigation, J.H.P., and J.W.J.; writing\u0026mdash;original draft, J.H.P. and J.W.J.; writing\u0026mdash;review \u0026amp; editing, J.H.P., J.W.J., T.Y.S., and H.W.C.; funding acquisition, T.Y.S. and H.W.C.; resources, J.H.P., J.W.J., S.W.J., Y.R.K., J.H.H., and G.R.N.; supervision, T.Y.S., and H.W.C. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe polysaccharides MP15R, T15, and T237 generated in this study are available from the corresponding author (Hyun Woo Choi, [email protected]) with a completed material transfer agreement. Any other data supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePost, M. J. Cultured meat from stem cells: Challenges and prospects. \u003cem\u003eMeat Sci.\u003c/em\u003e \u003cb\u003e92\u003c/b\u003e (3), 297\u0026ndash;301 (2012).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOgawa, M. et al. Edible mycelium as proliferation and differentiation support for anchorage-dependent animal cells in cultivated meat production. \u003cem\u003enpj Sci. Food\u003c/em\u003e. \u003cb\u003e8\u003c/b\u003e (1), 23 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGarrison, G. L., Biermacher, J. T. \u0026amp; Brorsen, B. W. How much will large-scale production of cell-cultured meat cost? \u003cem\u003eJ. Agric. Food Res.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 100358 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStout, A. J. et al. Engineered autocrine signaling eliminates muscle cell FGF2 requirements for cultured meat production. \u003cem\u003ebioRxiv\u003c/em\u003e (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePasitka, L. et al. Empirical economic analysis shows cost-effective continuous manufacturing of cultivated chicken using animal-free medium. \u003cem\u003eNat. Food\u003c/em\u003e. \u003cb\u003e5\u003c/b\u003e (8), 693\u0026ndash;702 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGuan, X., Zhou, J., Du, G. \u0026amp; Chen, J. Bioprocessing technology of muscle stem cells: implications for cultured meat. \u003cem\u003eTrends Biotechnol.\u003c/em\u003e \u003cb\u003e40\u003c/b\u003e (6), 721\u0026ndash;734 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLim, P. Y., Suntornnond, R. \u0026amp; Choudhury, D. The nutritional paradigm of cultivated meat: Bridging science and sustainability. \u003cem\u003eTrends Food Sci. Technol.\u003c/em\u003e \u003cb\u003e156\u003c/b\u003e, 104838 (2025).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYin, H., Price, F. \u0026amp; Rudnicki, M. A. Satellite Cells and the Muscle Stem Cell Niche. \u003cem\u003ePhysiol. Rev.\u003c/em\u003e \u003cb\u003e93\u003c/b\u003e (1), 23\u0026ndash;67 (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDumont, N. A., Wang, Y. X. \u0026amp; Rudnicki, M. A. Intrinsic and extrinsic mechanisms regulating satellite cell function. \u003cem\u003eDevelopment\u003c/em\u003e \u003cb\u003e142\u003c/b\u003e (9), 1572\u0026ndash;1581 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHung, M., Lo, H-F., Jones, G. E. L. \u0026amp; Krauss, R. S. The muscle stem cell niche at a glance. \u003cem\u003eJ. Cell Sci.\u003c/em\u003e ;\u003cb\u003e136\u003c/b\u003e(24). (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCharg\u0026Eacute;, S. B. P. \u0026amp; Rudnicki, M. A. Cellular and Molecular Regulation of Muscle Regeneration. \u003cem\u003ePhysiol. Rev.\u003c/em\u003e \u003cb\u003e84\u003c/b\u003e (1), 209\u0026ndash;238 (2004).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTsivitse, S. Notch and Wnt Signaling, Physiological Stimuli and Postnatal Myogenesis. \u003cem\u003eInt. J. Biol. Sci.\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e (3), 268\u0026ndash;281 (2010).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePasut, A. et al. Notch Signaling Rescues Loss of Satellite Cells Lacking Pax7 and Promotes Brown Adipogenic Differentiation. \u003cem\u003eCell. Rep.\u003c/em\u003e \u003cb\u003e16\u003c/b\u003e (2), 333\u0026ndash;343 (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFukada, S. Exercise/Resistance Training and Muscle Stem Cells. \u003cem\u003eenm\u003c/em\u003e \u003cb\u003e36\u003c/b\u003e (4), 737\u0026ndash;744 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSousa-Victor, P., Garc\u0026iacute;a-Prat, L., Serrano, A. L., Perdiguero, E. \u0026amp; Mu\u0026ntilde;oz-C\u0026aacute;noves, P. Muscle stem cell aging: regulation and rejuvenation. \u003cem\u003eTrends Endocrinol. Metabolism\u003c/em\u003e. \u003cb\u003e26\u003c/b\u003e (6), 287\u0026ndash;296 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eQin, G. et al. Notch signaling modulation enhances porcine muscle stem cell proliferation and differentiation. \u003cem\u003eBiochem. Biophys. Res. Commun.\u003c/em\u003e \u003cb\u003e752\u003c/b\u003e, 151456 (2025).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKolkmann, A. M., Van Essen, A., Post, M. J. \u0026amp; Moutsatsou, P. Development of a Chemically Defined Medium for in vitro Expansion of Primary Bovine Satellite Cells. \u003cem\u003eFront. Bioeng. Biotechnol.\u003c/em\u003e ;Volume 10\u0026ndash;2022. (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSkrivergaard, S. et al. A simple and robust serum-free media for the proliferation of muscle cells. \u003cem\u003eFood Res. Int.\u003c/em\u003e \u003cb\u003e172\u003c/b\u003e, 113194 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJudson, R. N. \u0026amp; Rossi, F. M. V. Towards stem cell therapies for skeletal muscle repair. \u003cem\u003enpj Regenerative Med.\u003c/em\u003e \u003cb\u003e5\u003c/b\u003e (1), 10 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBuchanan, S. M. et al. Pro-myogenic small molecules revealed by a chemical screen on primary muscle stem cells. \u003cem\u003eSkelet. Muscle\u003c/em\u003e. \u003cb\u003e10\u003c/b\u003e (1), 28 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRelaix, F. et al. Perspectives on skeletal muscle stem cells. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e (1), 692 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePang, K. T. et al. Insight into muscle stem cell regeneration and mechanobiology. \u003cem\u003eStem Cell Res. Ther.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e (1), 129 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu, S. et al. Species variations in muscle stem cell-mediated immunosuppression on T cells. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e (1), 23410 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen, H., Jia, Y. \u0026amp; Guo, Q. Chapter 6 - Polysaccharides and polysaccharide complexes as potential sources of antidiabetic compounds: A review. In: (ed Atta ur, R.) Studies in Natural Products Chemistry. 67: Elsevier; 199\u0026ndash;220. (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMahmoud, Y. A. G., El-Naggar, M. E., Abdel-Megeed, A. \u0026amp; El-Newehy, M. Recent Advancements in Microbial Polysaccharides: Synthesis and Applications. \u003cem\u003ePolymers-Basel\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 23 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhao, J. et al. Polysaccharide conjugate vaccine: A kind of vaccine with great development potential. \u003cem\u003eChin. Chem. Lett.\u003c/em\u003e \u003cb\u003e32\u003c/b\u003e (4), 1331\u0026ndash;1340 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShen, J. et al. Effect of Angelica polysaccharide on mouse myeloid-derived suppressor cells. \u003cem\u003eFront. Immunol.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 989230 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYe, R. et al. Eucommia ulmoides polysaccharide modified nano-selenium effectively alleviated DSS-induced colitis through enhancing intestinal mucosal barrier function and antioxidant capacity. \u003cem\u003eJ. Nanobiotechnol.\u003c/em\u003e \u003cb\u003e21\u003c/b\u003e (1), 222 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYuan, D., Li, C., Huang, Q., Fu, X. \u0026amp; Dong, H. Current advances in the anti-inflammatory effects and mechanisms of natural polysaccharides. \u003cem\u003eCrit. Rev. Food Sci. Nutr.\u003c/em\u003e \u003cb\u003e63\u003c/b\u003e (22), 5890\u0026ndash;5910 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee, J-H. et al. Effect of Crude Polysaccharides from Ecklonia cava Hydrolysate on Cell Proliferation and Differentiation of Hanwoo Muscle Stem Cells for Cultured Meat Production. \u003cem\u003eFoods\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e (4), 563 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShin, T. Y., Lee, M. R., Kim, J. C., Nai, Y. S. \u0026amp; Kim, J. S. A new strategy using entomopathogenic fungi for the control of tree borer insects. \u003cem\u003eEntomol. Res.\u003c/em\u003e \u003cb\u003e52\u003c/b\u003e (7), 327\u0026ndash;333 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNg, T. B. \u0026amp; Wang, H. X. Pharmacological actions of Cordyceps, a prized folk medicine. \u003cem\u003eJ. Pharm. Pharmacol.\u003c/em\u003e \u003cb\u003e57\u003c/b\u003e (12), 1509\u0026ndash;1519 (2005).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTuli, H. S., Sandhu, S. S. \u0026amp; Sharma, A. K. Pharmacological and therapeutic potential of Cordyceps with special reference to Cordycepin. \u003cem\u003e3 Biotech.\u003c/em\u003e \u003cb\u003e4\u003c/b\u003e (1), 1\u0026ndash;12 (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMantzoukas, S. \u0026amp; Eliopoulos, P. A. Endophytic Entomopathogenic Fungi: A Valuable Biological Control Tool against Plant Pests. \u003cem\u003eAppl. Sci-Basel\u003c/em\u003e ;\u003cb\u003e10\u003c/b\u003e(1). (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWu, S. Y. et al. Destruxin B Suppresses Drug-Resistant Colon Tumorigenesis and Stemness Is Associated with the Upregulation of miR-214 and Downregulation of mTOR/beta-Catenin Pathway. \u003cem\u003eCancers (Basel)\u003c/em\u003e ;\u003cb\u003e10\u003c/b\u003e(10). (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWu, N. et al. A review on polysaccharide biosynthesis in Cordyceps militaris. \u003cem\u003eInt. J. Biol. Macromol.\u003c/em\u003e \u003cb\u003e260\u003c/b\u003e (Pt 1), 129336 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen, L. S. et al. A polysaccharide from mycelia of Structural characterization, inhibition on α-glucosidase and improvement of insulin resistance in HepG2 cells. \u003cem\u003eProcess. Biochem.\u003c/em\u003e \u003cb\u003e125\u003c/b\u003e, 212\u0026ndash;221 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePark, Y., Choi, S., Kim, B. \u0026amp; Lee, S. G. Effects of Extracts on Macrophage as Immune Conductors. \u003cem\u003eAppl. Sci-Basel\u003c/em\u003e ;\u003cb\u003e11\u003c/b\u003e(5). (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYoon, S. Y., Lindroth, A. M., Kwon, S., Park, S. J. \u0026amp; Park, Y. J. Adenosine derivatives from Cordyceps exert antitumor effects against ovarian cancer cells through ENT1-mediated transport, induction of AMPK signaling, and consequent autophagic cell death. \u003cem\u003eBiomed. Pharmacother\u003c/em\u003e. \u003cb\u003e153\u003c/b\u003e, 113491 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChoi, S., Ferrari, G. \u0026amp; Tedesco, F. S. Cellular dynamics of myogenic cell migration: molecular mechanisms and implications for skeletal muscle cell therapies. \u003cem\u003eEMBO Mol. Med.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e (12), e12357 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGonzalez, M. L., Busse, N. I., Waits, C. M. \u0026amp; Johnson, S. E. Satellite cells and their regulation in livestock. \u003cem\u003eJ. Anim. Sci.\u003c/em\u003e \u003cb\u003e98\u003c/b\u003e (5), skaa081 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZygmunt, K., Otwinowska-Mindur, A., Pi\u0026oacute;rkowska, K. \u0026amp; Witarski, W. Influence of media composition on the level of bovine satellite cell proliferation. \u003cem\u003eAnimals\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e (11), 1855 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMatson, J. P. \u0026amp; Cook, J. G. Cell cycle proliferation decisions: the impact of single cell analyses. \u003cem\u003eFEBS J.\u003c/em\u003e \u003cb\u003e284\u003c/b\u003e (3), 362\u0026ndash;375 (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eQu, F., Guilak, F. \u0026amp; Mauck, R. L. Cell migration: implications for repair and regeneration in joint disease. \u003cem\u003eNat. Rev. Rheumatol.\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e (3), 167\u0026ndash;179 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDing, S. et al. Maintaining bovine satellite cells stemness through p38 pathway. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e (1), 10808 (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRastedt, W., Blumrich, E. M. \u0026amp; Dringen, R. Metabolism of Mannose in Cultured Primary Rat Neurons. \u003cem\u003eNeurochem Res.\u003c/em\u003e \u003cb\u003e42\u003c/b\u003e (8), 2282\u0026ndash;2293 (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang, H. H. et al. Nobiletin Prevents D-Galactose-Induced C2C12 Cell Aging by Improving Mitochondrial Function. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cb\u003e23\u003c/b\u003e, 19 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJang, J. W. \u003cem\u003eAntiviral activity of polysaccharide derived from entomopathogenic fungus\u003c/em\u003e (Jeonbuk National University, 2025).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim, J. K., Cho, M. L., Karnjanapratum, S., Shin, I. S. \u0026amp; You, S. G. In vitro and in vivo immunomodulatory activity of sulfated polysaccharides from Enteromorpha prolifera. \u003cem\u003eInt. J. Biol. Macromol.\u003c/em\u003e \u003cb\u003e49\u003c/b\u003e (5), 1051\u0026ndash;1058 (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim, H. S. et al. Activation of macrophages by polysaccharide isolated from Paecilomyces cicadae through toll-like receptor 4. \u003cem\u003eFood Chem. Toxicol.\u003c/em\u003e \u003cb\u003e50\u003c/b\u003e (9), 3190\u0026ndash;3197 (2012).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuang, L., Shen, M., Morris, G. A. \u0026amp; Xie, J. Sulfated polysaccharides: Immunomodulation and signaling mechanisms. \u003cem\u003eTrends Food Sci. Technol.\u003c/em\u003e \u003cb\u003e92\u003c/b\u003e, 1\u0026ndash;11 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee, D. Y. et al. The roles of media ingredients in muscle cell culture for cultured meat production-A mini-review. \u003cem\u003eFuture Foods\u003c/em\u003e ;\u003cb\u003e10\u003c/b\u003e. (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang, Y., Zou, L., Liu, W. \u0026amp; Chen, X. An overview of recent progress in engineering three-dimensional scaffolds for cultured meat production. \u003cem\u003eFoods\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e (13), 2614 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHe, H. et al. PDLIM5 affects chicken skeletal muscle satellite cell proliferation and differentiation via the p38-MAPK pathway. \u003cem\u003eAnimals\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e (4), 1016 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi, X. et al. Effect of IGF1 on Myogenic Proliferation and Differentiation of Bovine Skeletal Muscle Satellite Cells Through PI3K/AKT Signaling Pathway. \u003cem\u003eGenes\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e (12), 1494 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLong, X. Y., Yan, Q., Cai, L. J., Li, G. Y. \u0026amp; Luo, X. G. Box-Behnken design-based optimization for deproteinization of crude polysaccharides in berry residue using the Sevag method. \u003cem\u003eHeliyon\u003c/em\u003e ;\u003cb\u003e6\u003c/b\u003e(5). (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHan, J. H., Yu, J. S., Kim, D. H. \u0026amp; Choi, H. W. The characteristics of bovine satellite cells with highly scored genomic estimated breeding value. \u003cem\u003eJ. Anim. Reprod. Biotechnol.\u003c/em\u003e \u003cb\u003e38\u003c/b\u003e (3), 177\u0026ndash;187 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHan, J. H. et al. Comparative Analysis of Different Extracellular Matrices for the Maintenance of Bovine Satellite Cells. \u003cem\u003eAnimals\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e (23), 3496 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNo\u0026euml;, S. et al. The Myotube Analyzer: how to assess myogenic features in muscle stem cells. \u003cem\u003eSkelet. Muscle\u003c/em\u003e. \u003cb\u003e12\u003c/b\u003e (1), 12 (2022).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cultured meat, Bovine muscle stem cells, Polysaccharides, Cell proliferation, Myogenic differentiation","lastPublishedDoi":"10.21203/rs.3.rs-7935981/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7935981/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe finite proliferative lifespan and progressive loss of stemness in muscle stem cells present a significant bottleneck for the industrial-scale manufacturing of cultured meat. To overcome this limitation, we evaluated a microbial polysaccharide from \u003cem\u003eMetarhizium pinghaense\u003c/em\u003e 15R (MP15R) for its capacity to augment bovine satellite cell (BSC) functionality. Our \u003cem\u003ein vitro\u003c/em\u003e findings demonstrate that MP15R treatment enhanced BSC proliferation and migration while sustaining elevated expression of the canonical stem cell marker \u003cem\u003ePAX7\u003c/em\u003e throughout prolonged passaging. Moreover, upon induction, MP15R robustly promoted terminal myogenic differentiation, evidenced by the upregulation of key markers such as \u003cem\u003eMYOG\u003c/em\u003e and \u003cem\u003eMYHC\u003c/em\u003e and an increased myotube fusion index. Collectively, these results establish the MP15R polysaccharide as a sustainable, cost-effective bioactive agent with a bifunctional capacity to improve both the expansion and differentiation phases essential for cultured meat bioprocessing.\u003c/p\u003e","manuscriptTitle":"Polysaccharides from Metarhizium pinghaense: a novel biomaterial to modulate bovine satellite cell fate for cultured meat","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-17 12:49:02","doi":"10.21203/rs.3.rs-7935981/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-27T11:53:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-14T05:57:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"218099192380449202341421517214407223403","date":"2026-03-05T19:48:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8432328159329820296460382812843821668","date":"2026-03-03T16:11:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-19T15:44:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"97841982234269062320931417248419050887","date":"2026-02-09T10:47:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-05T14:49:05+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-27T16:59:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-24T13:05:04+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-24T13:03:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-10-24T02:10:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"462a3ad1-4612-4f1a-8362-b21a454784f9","owner":[],"postedDate":"November 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":58046426,"name":"Biological sciences/Biotechnology"},{"id":58046427,"name":"Biological sciences/Cell biology"},{"id":58046428,"name":"Biological sciences/Stem cells"}],"tags":[],"updatedAt":"2026-04-27T12:12:11+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-17 12:49:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7935981","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7935981","identity":"rs-7935981","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}