Muscle-type-dependent differences in satellite cell proliferation and differentiation in chick breast and leg muscles

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In this study, we compared the proliferation and differentiation characteristics of SCs derived from chick breast and leg muscles. In addition, the amino acid composition of each muscle tissue was analyzed to evaluate muscle- type-dependent differences. Breast and leg SCs were isolated and cultured, and their proliferative capacity was assessed by measuring cell number, population doubling time (PDT), and paired box 7 (Pax7) expression. Differentiation potential was evaluated using myogenin expression and myotube formation. Leg SCs maintained relatively stable cell numbers, PDT, and Pax7 expression across passages. In contrast, breast SCs showed a marked decline in cell number and Pax7 expression from passages 3 to 8. However, breast SCs exhibited relatively highly pronounced myogenin expression and myotube formation during early differentiation. Amino acid analysis revealed differences in the relative composition between breast and leg muscles, glutamic acid and glycine were abundant in both tissues. Collectively, these findings demonstrate muscle- type-dependent differences in SC proliferation and differentiation, accompanied by distinct tissue-level compositional profiles. By elucidating the intrinsic muscle type-dependent characteristics of SC proliferation and differentiation, this study provides foundational data for future research on muscle-specific meat quality improvement and cell culture strategies. satellite cells chick skeletal muscle proliferation differentiation Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Satellite cells (SCs), which reside between the basal lamina and sarcolemma of skeletal muscle fibers, are muscle stem cells that play a pivotal role in muscle regeneration following injury or trauma (Kaczmarek et al., 2021). During muscle injury, quiescent SCs are activated by crosstalk with various components of the muscle microenvironment, such as inflammatory cells, fibrogenic cells, and vasculature (Johnson et al., 2023). Activated SCs proliferate to supply a sufficient number of cells to the injury site, and a subset differentiates into myogenic precursor cells. Subsequently, they fuse with one another to form new myofibers or integrate into existing myofibers, thereby restoring the structural integrity of muscles (Yin et al., 2013). The process of proliferation and differentiation is a key regenerative function of SCs. The number and function of SCs are influenced by several factors. As aging progresses, SCs are significantly affected by environmental signals, leading to decreases in their number and function, which impairs muscle regeneration and exacerbates sarcopenia (Huo et al., 2022; Wiedmer et al., 2021). Furthermore, SCs exhibit different characteristics depending on the species, age, muscle fiber type, and anatomical location of origin. However, SCs are actively used in studies on muscle regeneration, therapy, and sarcopenia. Recent studies have focused on external conditions, such as culture temperature or media composition, to optimize the efficiency of in vitro proliferation and differentiation of SCs. SCs isolated from embryonic day 19 chicks have demonstrated superior proliferation and differentiation capabilities than those from 5-week-old chickens (Kim et al., 2024). Additionally, SCs isolated from the biceps femoris and pectoralis major of chicks show differential sensitivity to culture temperature (Harding et al., 2016). However, studies focusing solely on intrinsic differences in proliferation and differentiation capabilities of SCs based on the muscle type, excluding other variables, remain limited. In addition to cellular behavior, intrinsic metabolic features of each muscle type may contribute to their functional characteristics. Therefore, this study aimed to investigate the differences in proliferation and differentiation capabilities of SCs isolated from different chick muscle types and to compare the amino acid composition among tissues based on muscle types for an enhanced understanding of muscle- type-specific physiology and regenerative mechanisms. Materials and Methods Animals and housing All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Gyeongsang National University (IACUC No. GNU-250911-E0203). Seventeen embryonic-day- chicken embryos, randomly selected from the Leghorn breed regardless of sex, were used. SCs were isolated from tissue samples collected from the breast (pectoralis major) and legs (gastrocnemius, biceps femoris, and quadriceps femoris). Isolation and culture of SCs SCs were isolated from chick muscles as previously described, with slight modifications (Shahini et al., 2018). Tissues were minced into approximately 0.1-mm pieces using a scalpel. To 1 g tissue, 1 U collagenase D (Sigma-Aldrich, St. Louis, MO, USA), 2 U dispase II (Roche, Basel-Stadt, Switzerland), and 50 µL of 200 mM calcium chloride (Sigma-Aldrich) were added. The mixture was subjected to enzymatic dissociation at 37°C in a shaking incubator (LSI-3016R; DAIHAN LabTech, Namyangju, Republic of Korea) rotating at 200 rpm for approximately 40 min. The tissue homogenate was then sequentially filtered through cell strainers of 100, 70, and 40 µm (SPL, Pyeongtaek, Gyeonggi-do, Republic of Korea), followed by centrifugation at 675 rpm for 5 min (T05R; Hanil, Gimpo, Republic of Korea). The collected supernatant was centrifuged at 3,000 rpm for 5 min to obtain the pellet. To remove fibroblasts, the pellet was resuspended, plated in an uncoated cell culture dish (SPL), and incubated at 37°C for 5 min. The medium containing unattached SCs was collected for culture. Cells were maintained in a subconfluent state. For passaging, cells were detached using trypsin–ethylenediaminetetraacetic acid (Welgene, Gyeongsan, Republic of Korea) and centrifuged at 3,000 rpm for 5 min, and the pellet was resuspended in a growth medium [10% fetal bovine serum (Sigma-Aldrich), 1% penicillin/streptomycin (P/S; Sigma-Aldrich) in Ham's Nutrient Mixture F-10 (Welgene)]. To the resuspended pellet, 2 ng/mL basic fibroblast growth factor (Welgene) was added just before culture. To induce cell differentiation, the primary differentiation medium [5% horse serum (HS; Thermo Fisher Scientific, Waltham, MA, USA) and 1% P/S in Dulbecco's Modified Eagle Medium (DMEM; Cytiva, Marlborough, MA, USA)] was used to replace the growth medium for 2 days, followed by incubation with the secondary differentiation medium (10% HS and 1% P/S in DMEM), with medium changes every 2 days to promote differentiation. The detailed compositions of the culture media are summarized in Table 1 . Table 1 Composition of culture media used for proliferation and differentiation of SCs. Medium Formula Growth 89% Ham’s F-10, 10% FBS, 1% P/S + bFGF 2 ng/ml Primary differentiation 97% DMEM, 2% HS, 1% P/S Secondary differentiation 89% DMEM, 10% HS, 1% P/S Cell counting Cells were subcultured every 48 h and resuspended in 5 mL fresh growth medium. The cell suspension (10 µL) was mixed with 10 µL trypan blue (Thermo Fisher Scientific), and the number of viable cells was counted using a hemocytometer and phase-contrast microscope (EVOS M5000; Thermo Fisher Scientific). Determination of population doubling time (PDT) Cells were counted at each passage using trypan blue. PDT was calculated using the following formula: $$\:\text{P}\text{o}\text{p}\text{u}\text{l}\text{a}\text{t}\text{i}\text{o}\text{n}\:\text{d}\text{o}\text{u}\text{b}\text{l}\text{i}\text{n}\text{g}\:\text{t}\text{i}\text{m}\text{e}\:\left(h\right)=\frac{\text{dT}}{{\text{log}}_{\text{2}}\text{(}\frac{{\text{N}}_{\text{1}}\text{-}{\text{N}}_{\text{0}}}{{\text{N}}_{\text{0}}}\text{+1)}}$$ where dT is the incubation time, and N0 and N1 represent the initial and final cell numbers, respectively. Each passage was evaluated for cell number in triplicate. Immunofluorescence staining To determine the expression of the proliferation marker paired box 7 (Pax7), cells were stained after being subcultured and stabilized for 24 h. Staining was performed using an anti-Pax7 monoclonal mouse antibody (NKMAX, Seoul, Republic of Korea) as the primary antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG1 (Thermo Fisher Scientific) as the secondary antibody in triplicate wells. Cells were fixed at room temperature (RT, 20 ℃) with 4% paraformaldehyde (BYLABS, Hanam, Republic of Korea) for 20 min and subsequently washed twice with 1× phosphate-buffered saline (PBS; WELGENE, Namcheon, Republic of Korea) for 5 min each. Fixed cells were permeabilized with 0.2% Triton X-100 (Generay Biotech, Shanghai, China) at RT for 20 min and washed twice with 1× PBS for 5 min each. To block nonspecific binding, cells were incubated with 2% bovine serum albumin (HanLAB, Cheongju, Republic of Korea) in 1× PBS at RT for 30 min. Cells were incubated with the primary antibody diluted 1:500 in blocking buffer in the dark at 4°C overnight (12–14 h) and subsequently washed twice with 1× PBS. Then, cells were incubated with the secondary antibody diluted 1:1000 in blocking buffer in the dark at RT for 2 h. Subsequently, cells were washed twice with 1× PBS for 5 min each, and nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific) diluted 1:10000 in 1× PBS at RT for 5 min. Stained cells were imaged using an EVOS M5000 cell imaging system (Thermo Fisher Scientific). Images were processed using ImageJ (Rueden et al., 2017). To quantify Pax7 expression, five images were acquired from randomly selected locations within each well. The number of Hoechst-positive nuclei and Pax7-expressing cells were counted, and the ratio of Pax7-expressing cells to total nuclei was calculated. Similarly, to analyze the expression of the differentiation marker myogenin, cells were cultured in the differentiation medium for 24–48 h. Once myotubes were clearly visible, cells in triplicate wells were stained with a primary antibody against myogenin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and an Alexa Fluor 647-conjugated goat anti-mouse IgG2b secondary antibody (Thermo Fisher Scientific), following the same protocol described before. Statistical Analysis All assays were performed in triplicates. The results are presented as mean ± standard deviation. Statistical analyses were performed by analysis of variance using SPSS v.27.0 (Chicago, IL, USA), and differences between the mean values were calculated using Student–Newmann–Keuls and Tukey's range tests. Statistical significance was set at p < 0.05. Results and Discussion Proliferation of SCs Overall, changes in proliferative capacity during passaging showed distinct differences depending on the muscle of origin for SCs. The number of breast SCs significantly decreased from P3 to P8, with PDT showing an increase from P3 to P6 (Fig. 1 and Table 2 ). In contrast, leg SCs showed no significant changes in cell number or PDT with passaging, indicating that their proliferative capacity was stably maintained (Fig. 1 and Table 2 ). Therefore, breast SC exhibit a more pronounced decline in proliferative capacity with increasing passage number than leg SCs do. This finding is consistent with the general characteristics of SCs, with a limited number of divisions and decreased viability and proliferative capacity with continued passage (Chakravarthy et al., 2000). These findings suggest that breast SCs undergo passage-dependent senescence, whereas leg SCs retain proliferative stability across passages. This may reflect intrinsic fiber-type differences, as slow-twitch-derived SCs favor self-renewal over differentiation (Kalhovde et al., 2005; Motohashi et al., 2019). Following the removal of neural signals (exogenous factors) in adult mice, regeneration by SCs under identical slow stimulation results in 87% Type I MyHC expression in the soleus (slow-twitch muscle) compared to 13% in the extensor digitorum longus (fast-twitch muscle) (Kalhovde et al., 2005). Therefore, SCs possess intrinsically distinct myogenic programs depending on their muscle of origin. Chicken breast muscle is predominantly composed of white muscle (fast-twitch), whereas leg muscle consists mainly of red muscle (slow-twitch dominant) (Verdiglione et al., 2013). These differences in intrinsic characteristics support the distinct proliferation patterns observed in SCs derived from the breast (fast-twitch muscle) and legs (slow-twitch muscle) in the present study. SCs isolated from the pectoralis major of broilers undergo rapid myogenesis when cultured at 41°C, whereas the primitive population (stem cells) is maintained at 38°C, indicating that culture temperature is a critical exogenous factor determining cell fate (Gregg et al., 2023). In the present study, the culture temperature was 37°C, which was lower than the physiological temperature of chickens (41°C). Therefore, the culture condition might act as a factor affecting differentiation of the two SC populations, which differed in their dependence on oxidative metabolism, particularly during long-term culture (P8). Table 2 Comparison of from the numbers of SCs at different passages. Leg P3 P4 P5 P6 P7 P8 2.58 ± 0.63 2.67 ± 0.42 2.63 ± 0.21 2.59 ± 0.95 2.38 ± 0.54 2.43 ± 0.28 Breast 2.27 ± 0.46 a 2.02 ± 0.28 a 1.42 ± 0.3 b 0.88 ± 0.14 bc 0.88 ± 0.24 bc 0.67 ± 0.08 c The data represent the number of cells (× 10 6 ) and is presented as mean ± standard deviation. Values with different letters indicate significant differences ( p < 0.05). Muscle-type-dependent Pax7 expression in SCs Pax7 is a transcription factor, which promotes SC proliferation, and its decreased expression is a key determinant of their conversion into muscle cells during differentiation. In the present study, breast and leg SCs exhibited distinct passage-dependent patterns of Pax7 expression (Fig. 2 ). Breast SCs showed significantly decreased Pax7 expression at late passages, consistent with reduced cell numbers and increased PDT, suggesting an accelerated loss of stemness during passage. In contrast, leg SCs maintained stable Pax7 expression across passages, indicating sustained proliferative potential and resistance to passage-induced senescence. These differences reflect intrinsic fiber-type characteristics of the muscle of origin. SCs derived from slow- and fast-twitch muscles retain distinct phenotypic properties in vitro (Huang et al., 2006). Slow-twitch-derived cells exhibit a greater self-renewal capacity and lower differentiation potential than do fast-twitch-derived satellite cells (Motohashi et al., 2019). Consistent with these results, leg SCs (slow-twitch-derived SCs) maintained Pax7 expression during prolonged culture, whereas breast SCs (fast-twitch-derived SCs) showed an early decline, suggesting a tendency toward rapid activation and differentiation rather than long-term maintenance of stem cell-like properties. Muscle-type-dependent differentiation of SCs Myogenin is a key marker, which is expressed during SC differentiation, thereby inhibiting proliferation and inducing differentiation into muscle fibers (Zammit et al., 2017). Immunofluorescence analysis confirmed myogenin expression, and myotubes derived from breast SCs were relatively thick and distinct (Fig. 3 ). In summary, rapid myotube formation and relatively distinct myogenin expression patterns of breast SCs are indicative of a relatively high potential for early-stage differentiation. This result contrasts with our earlier observations in this study, where leg SCs relatively effectively maintained their proliferative capacity. Therefore, SCs may exhibit different proliferation and differentiation characteristics depending on their muscle of origin. Rapid differentiation and thick myotube formation observed for breast SCs suggest unique characteristics of fast-twitch SCs. Fast-twitch SCs exhibit stronger myogenic commitment than do slow-twitch SCs, and form relatively highly prominent and rapid myotubes upon induction of differentiation. Furthermore, fast-twitch cells preferentially commit to differentiation over self-renewal, leading to an early and rapid increase in the expression of differentiation factors (Motohashi et al., 2019). The fact that breast SCs exhibited relatively highly pronounced myogenin expression suggested that they possessed an intracellular program optimized for rapid myogenesis rather than proliferation. Amino acid composition of chick breast and leg muscles Fifteen amino acids were detected in both breast and leg muscle tissues. However, their relative abundance differed between the two muscle types (Fig. 4 ). Breast muscles contained relatively high levels of glutamic acid and glycine, whereas leg muscles contained relatively high levels of serine, alanine, glutamic acid, and glycine. Elevated alanine level in leg muscles may reflect its role in the glucose–alanine cycle that supplies energy for sustained activity and transports nitrogen to the liver (Petersen et al., 2019). This aligns with the metabolic demands of red muscle, which relies on oxidative metabolism for continuous movement of legs (Liu et al., 1996). Glutamic acid, which was abundant in both muscle types, is a major component of muscle proteins, and functions as an intermediate in the tricarboxylic acid cycle, highlighting its central role in energy metabolism (Brosnan, 2000). Similarly, high glycine levels likely reflect the abundance of collagen-rich connective tissue that supports muscle fibers, as glycine is a principal structural amino acid of collagen (Li et al., 2018). Overall, these amino acid profiles suggest that breast and leg muscles possess distinct metabolic adaptations that are consistent with their fiber-type compositions and physiological functions. Taken together, these findings suggest that SC behavior is associated with the intrinsic muscle- type-specific characteristics. Furthermore, the distinct amino acid profiles observed between the muscle types suggest coordinated differences at both the cellular and tissue levels. The present study was limited to characterizing the proliferation and differentiation phenotypes of chick satellite cells in vitro and did not elucidate the underlying molecular mechanisms or provide validation through in vivo models. Therefore, future research is warranted to verify key regulatory pathways using gene knockout techniques and to validate these findings within actual animal models of muscle regeneration. Declarations Ethics Approval All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Gyeongsang National University (IACUC No. GNU-250911-E0203). Author Contribution Conceptualization: A.Data curation: C, D, E, F.Formal analysis: C.Methodology: C, D.Validation: C, D, E, F.Investigation: C, D, E, F.Writing - original draft: C, B, A.Writing - review & editing: C, D, E, F, A, B. Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2023-00211920). This research was supported by the Regional Innovation System & Education (RISE) program through the RISE Center, Gyeongsangnam-do, funded by the Ministry of Education (MOE) and the Gyeongsangnam-do Provincial Government, Republic of Korea (2025-RISE-16-001). References Kaczmarek A, Kaczmarek M, Ciałowicz M, Clemente FM, Wolański P, Badicu G, Murawska-Ciałowicz E. 2021. The role of satellite cells in skeletal muscle regeneration—the effect of exercise and age. Biol 10:1056. Johnson AL, Kamal M, Parise G. 2023. The role of supporting cell populations in satellite cell mediated muscle repair. Cells 12:1968. Yin H, Price F, Rudnicki MA. 2013. Satellite cells and the muscle stem cell niche. Physiol Rev 93:23–67. Huo F, Liu Q, Liu H. 2022. Contribution of muscle satellite cells to sarcopenia. Front Physiol 13:892749. 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Chakravarthy MV, Davis BS, Booth FW. 2000. IGF-I restores satellite cell proliferative potential in immobilized old skeletal muscle. J Appl Physiol 89:1365–1379. Kalhovde JM, Jerkovic R, Sefland I, Cordonnier C, Calabria E, Schiaffino S, Lømo T. 2005. ‘Fast’ and ‘slow’ muscle fibres in hindlimb muscles of adult rats regenerate from intrinsically different satellite cells. J Physiol 562:847–857. Motohashi N, Uezumi A, Asakura A, Ikemoto-Uezumi M, Mori S, Mizunoe Y, Shigemoto K. 2019. Tbx1 regulates inherited metabolic and myogenic abilities of progenitor cells derived from slow-and fast-type muscle. Cell Death Differ 26:1024–1036. Verdiglione R, Cassandro M. 2013. Characterization of muscle fiber type in the pectoralis major muscle of slow-growing local and commercial chicken strains. Poult Sci 92:2433–2437. Gregg CR, Hutson BL, Flees JJ, Starkey CW, Starkey JD. 2023. Effect of standard and physiological cell culture temperatures on in vitro proliferation and differentiation of primary broiler chicken pectoralis major muscle satellite cells. Front Physiol 14:1288809. Huang YC, Dennis RG, Baar K. 2006. Cultured slow vs. fast skeletal muscle cells differ in physiology and responsiveness to stimulation. Am J Physiol Cell Physiol 291:C11-C17. Zammit PS. 2017. Function of the myogenic regulatory factors Myf5, MyoD, Myogenin and MRF4 in skeletal muscle, satellite cells and regenerative myogenesis. Semin Cell Dev Biol 72:19–32. Petersen KF, Dufour S, Cline GW, Shulman GI. 2019. Regulation of hepatic mitochondrial oxidation by glucose-alanine cycling during starvation in humans. J Clin Invest 129:4671–4675. Liu G, Xiong YL. 1996. Contribution of lipid and protein oxidation to rheological differences between chicken white and red muscle myofibrillar proteins. J Agric Food Chem 44:779–784. Brosnan JT. 2000. Glutamate, at the interface between amino acid and carbohydrate metabolism. J Nutr 130:988S-990S. Li P, Wu G. 2018. Roles of dietary glycine, proline, and hydroxyproline in collagen synthesis and animal growth. Amino Acids 50:29–38. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 12 Apr, 2026 Reviews received at journal 07 Apr, 2026 Reviewers agreed at journal 31 Mar, 2026 Reviewers agreed at journal 27 Mar, 2026 Reviewers invited by journal 25 Mar, 2026 Editor assigned by journal 18 Mar, 2026 Submission checks completed at journal 18 Mar, 2026 First submitted to journal 17 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9147387","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":611752115,"identity":"8dd92779-fe7b-441d-9288-dc23d6725fb1","order_by":0,"name":"Hyo Jin Lee","email":"","orcid":"","institution":"Gyeongsang National University","correspondingAuthor":false,"prefix":"","firstName":"Hyo","middleName":"Jin","lastName":"Lee","suffix":""},{"id":611752116,"identity":"760ac730-f175-4e58-ba99-6f98385366de","order_by":1,"name":"Dong Bin Kim","email":"","orcid":"","institution":"Gyeongsang National University","correspondingAuthor":false,"prefix":"","firstName":"Dong","middleName":"Bin","lastName":"Kim","suffix":""},{"id":611752119,"identity":"47dbccd9-1d73-4bc6-a3f5-3f079d91c02a","order_by":2,"name":"Hye Won Lee","email":"","orcid":"","institution":"Gyeongsang National University","correspondingAuthor":false,"prefix":"","firstName":"Hye","middleName":"Won","lastName":"Lee","suffix":""},{"id":611752121,"identity":"a90ff758-b55e-43a7-9c48-b84f711ba776","order_by":3,"name":"Ho Gun Jang","email":"","orcid":"","institution":"Gyeongsang National University","correspondingAuthor":false,"prefix":"","firstName":"Ho","middleName":"Gun","lastName":"Jang","suffix":""},{"id":611752124,"identity":"7b7fff5e-abb9-4859-8d57-9ecf15c4f8ca","order_by":4,"name":"Jong Hyuk Kim","email":"","orcid":"","institution":"Chungbuk National University","correspondingAuthor":false,"prefix":"","firstName":"Jong","middleName":"Hyuk","lastName":"Kim","suffix":""},{"id":611752125,"identity":"36b792a1-527b-4ff7-a66a-cf4a3763f055","order_by":5,"name":"Seung Yun Lee","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYFAC5gYGxj//5WBcAyK0MAK1NDAbk64lsYFoLfIzEhsffNzBlj4/useA4UcNg7F5AwEtBjcSmw1nnuHJ3XjnjAFjzzEGM5kDhLRIJLZJ87BJ5G6ckWPAwNvAYCNBhMPaf/9hM0g3BGph/EuMFoYbiW3MjG0JCfISOQbMQFvMCGoxOPOwWbLnzAHDDRJpBYdljkkYE3ZYe/LBDz8qDsjLz0je+PBNjY3hDIIOE0iAWneAgQGICPuEgYH/ANS6BiIUj4JRMApGwcgEABpYPtN1H+MLAAAAAElFTkSuQmCC","orcid":"","institution":"Gyeongsang National University","correspondingAuthor":true,"prefix":"","firstName":"Seung","middleName":"Yun","lastName":"Lee","suffix":""}],"badges":[],"createdAt":"2026-03-17 10:11:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9147387/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9147387/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105446462,"identity":"3a172515-0454-4e79-be16-2f409650fe5a","added_by":"auto","created_at":"2026-03-26 07:17:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":144237,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of the proliferative capacity of SCs between leg and breast groups.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Cell numbers and (B) population doubling time (PDT) measured from P3 to P8. Data are presented as the mean ± standard deviation of three independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. TMT, too much doubling time.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9147387/v1/21e1d3842c890bb371bf5a33.png"},{"id":105446464,"identity":"ef427f94-304f-443c-9357-06f002ee099f","added_by":"auto","created_at":"2026-03-26 07:17:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2764939,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of Pax7 expression in SCs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative immunofluorescence images of cells at early and late passages. Blue and green indicate nuclei and Pax7, respectively. (B) Quantification of Pax7-positive cell ratio. (C, D) Comparison of the Pax7 ratio between early and late passages within (C) leg and (D) breast cell populations. Data are presented as mean ± standard deviation. *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9147387/v1/27877134d71195698c09a1d8.png"},{"id":105446465,"identity":"d4b6f6f5-c4c6-4b8c-89ae-f68d689e7e93","added_by":"auto","created_at":"2026-03-26 07:17:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4576700,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of myogenic differentiation between SCs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative immunofluorescence images of differentiated cells. Myogenin is shown in green, and nuclei are shown in blue.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9147387/v1/7823be4e9c039b8cbe29df97.png"},{"id":105446463,"identity":"cd932c0f-1307-432c-89f5-3e02e48f370f","added_by":"auto","created_at":"2026-03-26 07:17:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":227319,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeatmap analysis of amino acid composition in leg and breast muscles.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe color scale on the right indicates composition level, in which dark blue represents relatively high abundance, and light yellow represents relatively low abundance.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9147387/v1/0b6d41b37c852cc539074e09.png"},{"id":105566280,"identity":"7a848a96-ef6f-4aef-be09-f013260a2cc4","added_by":"auto","created_at":"2026-03-27 12:56:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10305466,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9147387/v1/ab6a0f72-b277-4511-bca7-ae49a316fc68.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Muscle-type-dependent differences in satellite cell proliferation and differentiation in chick breast and leg muscles","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSatellite cells (SCs), which reside between the basal lamina and sarcolemma of skeletal muscle fibers, are muscle stem cells that play a pivotal role in muscle regeneration following injury or trauma (Kaczmarek et al., 2021). During muscle injury, quiescent SCs are activated by crosstalk with various components of the muscle microenvironment, such as inflammatory cells, fibrogenic cells, and vasculature (Johnson et al., 2023). Activated SCs proliferate to supply a sufficient number of cells to the injury site, and a subset differentiates into myogenic precursor cells. Subsequently, they fuse with one another to form new myofibers or integrate into existing myofibers, thereby restoring the structural integrity of muscles (Yin et al., 2013). The process of proliferation and differentiation is a key regenerative function of SCs.\u003c/p\u003e \u003cp\u003eThe number and function of SCs are influenced by several factors. As aging progresses, SCs are significantly affected by environmental signals, leading to decreases in their number and function, which impairs muscle regeneration and exacerbates sarcopenia (Huo et al., 2022; Wiedmer et al., 2021). Furthermore, SCs exhibit different characteristics depending on the species, age, muscle fiber type, and anatomical location of origin. However, SCs are actively used in studies on muscle regeneration, therapy, and sarcopenia. Recent studies have focused on external conditions, such as culture temperature or media composition, to optimize the efficiency of \u003cem\u003ein vitro\u003c/em\u003e proliferation and differentiation of SCs. SCs isolated from embryonic day 19 chicks have demonstrated superior proliferation and differentiation capabilities than those from 5-week-old chickens (Kim et al., 2024). Additionally, SCs isolated from the biceps femoris and pectoralis major of chicks show differential sensitivity to culture temperature (Harding et al., 2016). However, studies focusing solely on intrinsic differences in proliferation and differentiation capabilities of SCs based on the muscle type, excluding other variables, remain limited. In addition to cellular behavior, intrinsic metabolic features of each muscle type may contribute to their functional characteristics. Therefore, this study aimed to investigate the differences in proliferation and differentiation capabilities of SCs isolated from different chick muscle types and to compare the amino acid composition among tissues based on muscle types for an enhanced understanding of muscle- type-specific physiology and regenerative mechanisms.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and housing\u003c/h2\u003e \u003cp\u003e All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Gyeongsang National University (IACUC No. GNU-250911-E0203). Seventeen embryonic-day- chicken embryos, randomly selected from the Leghorn breed regardless of sex, were used. SCs were isolated from tissue samples collected from the breast (pectoralis major) and legs (gastrocnemius, biceps femoris, and quadriceps femoris).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIsolation and culture of SCs\u003c/h3\u003e\n\u003cp\u003eSCs were isolated from chick muscles as previously described, with slight modifications (Shahini et al., 2018). Tissues were minced into approximately 0.1-mm pieces using a scalpel. To 1 g tissue, 1 U collagenase D (Sigma-Aldrich, St. Louis, MO, USA), 2 U dispase II (Roche, Basel-Stadt, Switzerland), and 50 \u0026micro;L of 200 mM calcium chloride (Sigma-Aldrich) were added. The mixture was subjected to enzymatic dissociation at 37\u0026deg;C in a shaking incubator (LSI-3016R; DAIHAN LabTech, Namyangju, Republic of Korea) rotating at 200 rpm for approximately 40 min. The tissue homogenate was then sequentially filtered through cell strainers of 100, 70, and 40 \u0026micro;m (SPL, Pyeongtaek, Gyeonggi-do, Republic of Korea), followed by centrifugation at 675 rpm for 5 min (T05R; Hanil, Gimpo, Republic of Korea). The collected supernatant was centrifuged at 3,000 rpm for 5 min to obtain the pellet. To remove fibroblasts, the pellet was resuspended, plated in an uncoated cell culture dish (SPL), and incubated at 37\u0026deg;C for 5 min. The medium containing unattached SCs was collected for culture. Cells were maintained in a subconfluent state. For passaging, cells were detached using trypsin\u0026ndash;ethylenediaminetetraacetic acid (Welgene, Gyeongsan, Republic of Korea) and centrifuged at 3,000 rpm for 5 min, and the pellet was resuspended in a growth medium [10% fetal bovine serum (Sigma-Aldrich), 1% penicillin/streptomycin (P/S; Sigma-Aldrich) in Ham's Nutrient Mixture F-10 (Welgene)]. To the resuspended pellet, 2 ng/mL basic fibroblast growth factor (Welgene) was added just before culture. To induce cell differentiation, the primary differentiation medium [5% horse serum (HS; Thermo Fisher Scientific, Waltham, MA, USA) and 1% P/S in Dulbecco's Modified Eagle Medium (DMEM; Cytiva, Marlborough, MA, USA)] was used to replace the growth medium for 2 days, followed by incubation with the secondary differentiation medium (10% HS and 1% P/S in DMEM), with medium changes every 2 days to promote differentiation. The detailed compositions of the culture media are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComposition of culture media used for proliferation and differentiation of SCs.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMedium\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFormula\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGrowth\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e89% Ham\u0026rsquo;s F-10, 10% FBS, 1% P/S\u0026thinsp;+\u0026thinsp;bFGF 2 ng/ml\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePrimary differentiation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e97% DMEM, 2% HS, 1% P/S\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSecondary differentiation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e89% DMEM, 10% HS, 1% P/S\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eCell counting\u003c/h3\u003e\n\u003cp\u003eCells were subcultured every 48 h and resuspended in 5 mL fresh growth medium. The cell suspension (10 \u0026micro;L) was mixed with 10 \u0026micro;L trypan blue (Thermo Fisher Scientific), and the number of viable cells was counted using a hemocytometer and phase-contrast microscope (EVOS M5000; Thermo Fisher Scientific).\u003c/p\u003e \u003cp\u003eDetermination of population doubling time (PDT)\u003c/p\u003e \u003cp\u003eCells were counted at each passage using trypan blue. PDT was calculated using the following formula:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{P}\\text{o}\\text{p}\\text{u}\\text{l}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n}\\:\\text{d}\\text{o}\\text{u}\\text{b}\\text{l}\\text{i}\\text{n}\\text{g}\\:\\text{t}\\text{i}\\text{m}\\text{e}\\:\\left(h\\right)=\\frac{\\text{dT}}{{\\text{log}}_{\\text{2}}\\text{(}\\frac{{\\text{N}}_{\\text{1}}\\text{-}{\\text{N}}_{\\text{0}}}{{\\text{N}}_{\\text{0}}}\\text{+1)}}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003edT\u003c/em\u003e is the incubation time, and \u003cem\u003eN0\u003c/em\u003e and \u003cem\u003eN1\u003c/em\u003e represent the initial and final cell numbers, respectively. Each passage was evaluated for cell number in triplicate.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence staining\u003c/h3\u003e\n\u003cp\u003eTo determine the expression of the proliferation marker paired box 7 (Pax7), cells were stained after being subcultured and stabilized for 24 h. Staining was performed using an anti-Pax7 monoclonal mouse antibody (NKMAX, Seoul, Republic of Korea) as the primary antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG1 (Thermo Fisher Scientific) as the secondary antibody in triplicate wells. Cells were fixed at room temperature (RT, 20 ℃) with 4% paraformaldehyde (BYLABS, Hanam, Republic of Korea) for 20 min and subsequently washed twice with 1\u0026times; phosphate-buffered saline (PBS; WELGENE, Namcheon, Republic of Korea) for 5 min each. Fixed cells were permeabilized with 0.2% Triton X-100 (Generay Biotech, Shanghai, China) at RT for 20 min and washed twice with 1\u0026times; PBS for 5 min each. To block nonspecific binding, cells were incubated with 2% bovine serum albumin (HanLAB, Cheongju, Republic of Korea) in 1\u0026times; PBS at RT for 30 min. Cells were incubated with the primary antibody diluted 1:500 in blocking buffer in the dark at 4\u0026deg;C overnight (12\u0026ndash;14 h) and subsequently washed twice with 1\u0026times; PBS. Then, cells were incubated with the secondary antibody diluted 1:1000 in blocking buffer in the dark at RT for 2 h. Subsequently, cells were washed twice with 1\u0026times; PBS for 5 min each, and nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific) diluted 1:10000 in 1\u0026times; PBS at RT for 5 min. Stained cells were imaged using an EVOS M5000 cell imaging system (Thermo Fisher Scientific). Images were processed using ImageJ (Rueden et al., 2017). To quantify Pax7 expression, five images were acquired from randomly selected locations within each well. The number of Hoechst-positive nuclei and Pax7-expressing cells were counted, and the ratio of Pax7-expressing cells to total nuclei was calculated. Similarly, to analyze the expression of the differentiation marker myogenin, cells were cultured in the differentiation medium for 24\u0026ndash;48 h. Once myotubes were clearly visible, cells in triplicate wells were stained with a primary antibody against myogenin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and an Alexa Fluor 647-conjugated goat anti-mouse IgG2b secondary antibody (Thermo Fisher Scientific), following the same protocol described before.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll assays were performed in triplicates. The results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical analyses were performed by analysis of variance using SPSS v.27.0 (Chicago, IL, USA), and differences between the mean values were calculated using Student\u0026ndash;Newmann\u0026ndash;Keuls and Tukey's range tests. Statistical significance was set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eProliferation of SCs\u003c/h2\u003e \u003cp\u003eOverall, changes in proliferative capacity during passaging showed distinct differences depending on the muscle of origin for SCs. The number of breast SCs significantly decreased from P3 to P8, with PDT showing an increase from P3 to P6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In contrast, leg SCs showed no significant changes in cell number or PDT with passaging, indicating that their proliferative capacity was stably maintained (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Therefore, breast SC exhibit a more pronounced decline in proliferative capacity with increasing passage number than leg SCs do. This finding is consistent with the general characteristics of SCs, with a limited number of divisions and decreased viability and proliferative capacity with continued passage (Chakravarthy et al., 2000). These findings suggest that breast SCs undergo passage-dependent senescence, whereas leg SCs retain proliferative stability across passages. This may reflect intrinsic fiber-type differences, as slow-twitch-derived SCs favor self-renewal over differentiation (Kalhovde et al., 2005; Motohashi et al., 2019). Following the removal of neural signals (exogenous factors) in adult mice, regeneration by SCs under identical slow stimulation results in 87% Type I MyHC expression in the soleus (slow-twitch muscle) compared to 13% in the extensor digitorum longus (fast-twitch muscle) (Kalhovde et al., 2005). Therefore, SCs possess intrinsically distinct myogenic programs depending on their muscle of origin. Chicken breast muscle is predominantly composed of white muscle (fast-twitch), whereas leg muscle consists mainly of red muscle (slow-twitch dominant) (Verdiglione et al., 2013). These differences in intrinsic characteristics support the distinct proliferation patterns observed in SCs derived from the breast (fast-twitch muscle) and legs (slow-twitch muscle) in the present study. SCs isolated from the pectoralis major of broilers undergo rapid myogenesis when cultured at 41\u0026deg;C, whereas the primitive population (stem cells) is maintained at 38\u0026deg;C, indicating that culture temperature is a critical exogenous factor determining cell fate (Gregg et al., 2023). In the present study, the culture temperature was 37\u0026deg;C, which was lower than the physiological temperature of chickens (41\u0026deg;C). Therefore, the culture condition might act as a factor affecting differentiation of the two SC populations, which differed in their dependence on oxidative metabolism, particularly during long-term culture (P8).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of from the numbers of SCs at different passages.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eLeg\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP6\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eP8\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBreast\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eThe data represent the number of cells (\u0026times; 10\u003csup\u003e6\u003c/sup\u003e) and is presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Values with different letters indicate significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMuscle-type-dependent Pax7 expression in SCs\u003c/h3\u003e\n\u003cp\u003ePax7 is a transcription factor, which promotes SC proliferation, and its decreased expression is a key determinant of their conversion into muscle cells during differentiation. In the present study, breast and leg SCs exhibited distinct passage-dependent patterns of Pax7 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Breast SCs showed significantly decreased Pax7 expression at late passages, consistent with reduced cell numbers and increased PDT, suggesting an accelerated loss of stemness during passage. In contrast, leg SCs maintained stable Pax7 expression across passages, indicating sustained proliferative potential and resistance to passage-induced senescence. These differences reflect intrinsic fiber-type characteristics of the muscle of origin. SCs derived from slow- and fast-twitch muscles retain distinct phenotypic properties \u003cem\u003ein vitro\u003c/em\u003e (Huang et al., 2006). Slow-twitch-derived cells exhibit a greater self-renewal capacity and lower differentiation potential than do fast-twitch-derived satellite cells (Motohashi et al., 2019). Consistent with these results, leg SCs (slow-twitch-derived SCs) maintained Pax7 expression during prolonged culture, whereas breast SCs (fast-twitch-derived SCs) showed an early decline, suggesting a tendency toward rapid activation and differentiation rather than long-term maintenance of stem cell-like properties.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMuscle-type-dependent differentiation of SCs\u003c/h2\u003e \u003cp\u003eMyogenin is a key marker, which is expressed during SC differentiation, thereby inhibiting proliferation and inducing differentiation into muscle fibers (Zammit et al., 2017). Immunofluorescence analysis confirmed myogenin expression, and myotubes derived from breast SCs were relatively thick and distinct (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In summary, rapid myotube formation and relatively distinct myogenin expression patterns of breast SCs are indicative of a relatively high potential for early-stage differentiation. This result contrasts with our earlier observations in this study, where leg SCs relatively effectively maintained their proliferative capacity. Therefore, SCs may exhibit different proliferation and differentiation characteristics depending on their muscle of origin. Rapid differentiation and thick myotube formation observed for breast SCs suggest unique characteristics of fast-twitch SCs. Fast-twitch SCs exhibit stronger myogenic commitment than do slow-twitch SCs, and form relatively highly prominent and rapid myotubes upon induction of differentiation. Furthermore, fast-twitch cells preferentially commit to differentiation over self-renewal, leading to an early and rapid increase in the expression of differentiation factors (Motohashi et al., 2019). The fact that breast SCs exhibited relatively highly pronounced myogenin expression suggested that they possessed an intracellular program optimized for rapid myogenesis rather than proliferation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAmino acid composition of chick breast and leg muscles\u003c/h2\u003e \u003cp\u003eFifteen amino acids were detected in both breast and leg muscle tissues. However, their relative abundance differed between the two muscle types (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Breast muscles contained relatively high levels of glutamic acid and glycine, whereas leg muscles contained relatively high levels of serine, alanine, glutamic acid, and glycine. Elevated alanine level in leg muscles may reflect its role in the glucose\u0026ndash;alanine cycle that supplies energy for sustained activity and transports nitrogen to the liver (Petersen et al., 2019). This aligns with the metabolic demands of red muscle, which relies on oxidative metabolism for continuous movement of legs (Liu et al., 1996). Glutamic acid, which was abundant in both muscle types, is a major component of muscle proteins, and functions as an intermediate in the tricarboxylic acid cycle, highlighting its central role in energy metabolism (Brosnan, 2000). Similarly, high glycine levels likely reflect the abundance of collagen-rich connective tissue that supports muscle fibers, as glycine is a principal structural amino acid of collagen (Li et al., 2018). Overall, these amino acid profiles suggest that breast and leg muscles possess distinct metabolic adaptations that are consistent with their fiber-type compositions and physiological functions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTaken together, these findings suggest that SC behavior is associated with the intrinsic muscle- type-specific characteristics. Furthermore, the distinct amino acid profiles observed between the muscle types suggest coordinated differences at both the cellular and tissue levels. The present study was limited to characterizing the proliferation and differentiation phenotypes of chick satellite cells \u003cem\u003ein vitro\u003c/em\u003e and did not elucidate the underlying molecular mechanisms or provide validation through \u003cem\u003ein vivo\u003c/em\u003e models. Therefore, future research is warranted to verify key regulatory pathways using gene knockout techniques and to validate these findings within actual animal models of muscle regeneration.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics Approval\u003c/h2\u003e \u003cp\u003eAll experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Gyeongsang National University (IACUC No. GNU-250911-E0203).\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: A.Data curation: C, D, E, F.Formal analysis: C.Methodology: C, D.Validation: C, D, E, F.Investigation: C, D, E, F.Writing - original draft: C, B, A.Writing - review \u0026amp; editing: C, D, E, F, A, B.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2023-00211920). This research was supported by the Regional Innovation System \u0026amp; Education (RISE) program through the RISE Center, Gyeongsangnam-do, funded by the Ministry of Education (MOE) and the Gyeongsangnam-do Provincial Government, Republic of Korea (2025-RISE-16-001).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e Kaczmarek A, Kaczmarek M, Ciałowicz M, Clemente FM, Wolański P, Badicu G, Murawska-Ciałowicz E. 2021. The role of satellite cells in skeletal muscle regeneration\u0026mdash;the effect of exercise and age. Biol 10:1056.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Johnson AL, Kamal M, Parise G. 2023. The role of supporting cell populations in satellite cell mediated muscle repair. Cells 12:1968.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Yin H, Price F, Rudnicki MA. 2013. Satellite cells and the muscle stem cell niche. Physiol Rev 93:23\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Huo F, Liu Q, Liu H. 2022. Contribution of muscle satellite cells to sarcopenia. Front Physiol 13:892749.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Wiedmer P, Jung T, Castro JP, Pomatto LC, Sun PY, Davies KJ, Grune T. 2021. Sarcopenia\u0026ndash;Molecular mechanisms and open questions. Ageing Res Rev 65:101200.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Kim CJ, Kim SH, Lee EY, Hwang YH, Lee SY, Joo ST. 2024. Effect of chicken age on proliferation and differentiation abilities of muscle stem cells and nutritional characteristics of cultured meat tissue. Food Sci Anim Resour 44:1167.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Harding RL, Halevy O, Yahav S, Velleman SG. 2016. The effect of temperature on proliferation and differentiation of chicken skeletal muscle satellite cells isolated from different muscle types. Physiol Rep 4:e12770.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Shahini A, Vydiam K, Choudhury D, Rajabian N, Nguyen T, Lei P, Andreadis ST. 2018. Efficient and high yield isolation of myoblasts from skeletal muscle. Stem Cell Res 30:122\u0026ndash;129.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Rueden CT, Schindelin J, Hiner MC, DeZonia BE, Walter AE, Arena ET, Eliceiri KW. 2017. ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinformatics 18:529.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Chakravarthy MV, Davis BS, Booth FW. 2000. IGF-I restores satellite cell proliferative potential in immobilized old skeletal muscle. J Appl Physiol 89:1365\u0026ndash;1379.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Kalhovde JM, Jerkovic R, Sefland I, Cordonnier C, Calabria E, Schiaffino S, L\u0026oslash;mo T. 2005. \u0026lsquo;Fast\u0026rsquo; and \u0026lsquo;slow\u0026rsquo; muscle fibres in hindlimb muscles of adult rats regenerate from intrinsically different satellite cells. J Physiol 562:847\u0026ndash;857.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Motohashi N, Uezumi A, Asakura A, Ikemoto-Uezumi M, Mori S, Mizunoe Y, Shigemoto K. 2019. Tbx1 regulates inherited metabolic and myogenic abilities of progenitor cells derived from slow-and fast-type muscle. Cell Death Differ 26:1024\u0026ndash;1036.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Verdiglione R, Cassandro M. 2013. Characterization of muscle fiber type in the pectoralis major muscle of slow-growing local and commercial chicken strains. Poult Sci 92:2433\u0026ndash;2437.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Gregg CR, Hutson BL, Flees JJ, Starkey CW, Starkey JD. 2023. Effect of standard and physiological cell culture temperatures on in vitro proliferation and differentiation of primary broiler chicken pectoralis major muscle satellite cells. Front Physiol 14:1288809.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Huang YC, Dennis RG, Baar K. 2006. Cultured slow vs. fast skeletal muscle cells differ in physiology and responsiveness to stimulation. Am J Physiol Cell Physiol 291:C11-C17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Zammit PS. 2017. Function of the myogenic regulatory factors Myf5, MyoD, Myogenin and MRF4 in skeletal muscle, satellite cells and regenerative myogenesis. Semin Cell Dev Biol 72:19\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Petersen KF, Dufour S, Cline GW, Shulman GI. 2019. Regulation of hepatic mitochondrial oxidation by glucose-alanine cycling during starvation in humans. J Clin Invest 129:4671\u0026ndash;4675.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Liu G, Xiong YL. 1996. Contribution of lipid and protein oxidation to rheological differences between chicken white and red muscle myofibrillar proteins. J Agric Food Chem 44:779\u0026ndash;784.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Brosnan JT. 2000. Glutamate, at the interface between amino acid and carbohydrate metabolism. J Nutr 130:988S-990S.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Li P, Wu G. 2018. Roles of dietary glycine, proline, and hydroxyproline in collagen synthesis and animal growth. Amino Acids 50:29\u0026ndash;38.\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":"food-science-of-animal-resources","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food Science of Animal Resources](https://link.springer.com/journal/44463)","snPcode":"44463","submissionUrl":"https://submission.springernature.com/new-submission/44463/3?","title":"Food Science of Animal Resources","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"satellite cells, chick, skeletal muscle, proliferation, differentiation","lastPublishedDoi":"10.21203/rs.3.rs-9147387/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9147387/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"In chickens, studies focusing exclusively on intrinsic muscle type-dependent differences in proliferation and differentiation of satellite cells (SCs) are scanty. In this study, we compared the proliferation and differentiation characteristics of SCs derived from chick breast and leg muscles. In addition, the amino acid composition of each muscle tissue was analyzed to evaluate muscle- type-dependent differences. Breast and leg SCs were isolated and cultured, and their proliferative capacity was assessed by measuring cell number, population doubling time (PDT), and paired box 7 (Pax7) expression. Differentiation potential was evaluated using myogenin expression and myotube formation. Leg SCs maintained relatively stable cell numbers, PDT, and Pax7 expression across passages. In contrast, breast SCs showed a marked decline in cell number and Pax7 expression from passages 3 to 8. However, breast SCs exhibited relatively highly pronounced myogenin expression and myotube formation during early differentiation. Amino acid analysis revealed differences in the relative composition between breast and leg muscles, glutamic acid and glycine were abundant in both tissues. Collectively, these findings demonstrate muscle- type-dependent differences in SC proliferation and differentiation, accompanied by distinct tissue-level compositional profiles. By elucidating the intrinsic muscle type-dependent characteristics of SC proliferation and differentiation, this study provides foundational data for future research on muscle-specific meat quality improvement and cell culture strategies.","manuscriptTitle":"Muscle-type-dependent differences in satellite cell proliferation and differentiation in chick breast and leg muscles","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-26 07:17:03","doi":"10.21203/rs.3.rs-9147387/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-12T15:04:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-08T02:19:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"114623105638573497038699603153256484411","date":"2026-03-31T05:03:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"20998482519058175625793746294536416744","date":"2026-03-27T05:49:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-25T05:35:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-18T23:45:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-18T09:31:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Food Science of Animal Resources","date":"2026-03-17T09:58:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"food-science-of-animal-resources","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food Science of Animal Resources](https://link.springer.com/journal/44463)","snPcode":"44463","submissionUrl":"https://submission.springernature.com/new-submission/44463/3?","title":"Food Science of Animal Resources","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0a7fb9f5-c9ca-4a98-82f6-2783be7f86d9","owner":[],"postedDate":"March 26th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-26T07:17:03+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-26 07:17:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9147387","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9147387","identity":"rs-9147387","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}