Chicken Satellite Cells Research Articles

Chicken Embryo Fibroblast Viability and Trans-Differentiation Potential for Cultured Meat Production Across Passages.

This study was conducted to analyze the viability of primary chicken embryo fibroblasts and the efficiency of adipogenic trans-differentiation for cultured meat production. In isolating chicken embryo fibroblasts (CEFs) from a heterogeneous cell pool containing chicken satellite cells (CSCs), over 90% of CEFs expressed CD29 and vimentin. The analysis of the proliferative capabilities of CEFs revealed no significant differences in EdU-positive cells (%), cumulative cell number, doubling time, and growth rate from passage 1 to passage 9 (p > 0.05). This indicates that CEFs can be isolated by 2 h of pre-plating and survive stably up to passage 9, and that primary fibroblasts can serve as a valuable cell source for the cultured meat industry. Adipogenic trans-differentiation was induced up to passage 9 of CEFs. As passages increased, lipid accumulation and adipocyte size significantly decreased (p < 0.05). The reduced differentiation rate of primary CEFs with increasing passages poses a major challenge to the cost and efficiency of cultured meat production. Thus, effective cell management and the maintenance of cellular characteristics for a long time are crucial for ensuring stable and efficient cultured fat production in the cultured meat industry.

Trypsin-treated chickpea protein hydrolysate enhances the cytoaffinity of microbeads for cultured meat application

Cultured meat is believed to be a promising alternative to conventional meat production that can reduce environmental impacts, animal suffering, and food safety risks. However, one of the major challenges in producing cultured meat is to provide suitable microcarriers that can support cell attachment, proliferation, and differentiation. In this study, we developed novel microcarriers based on chickpea protein hydrolysates functionalized with trypsin. These microcarriers exhibited superior cytoaffinity and proliferation for various types of cultured cells, including C2C12, porcine myoblasts, chicken satellite cells, and 3T3-L1. Moreover, these microcarriers enabled cell differentiation into muscle or fat cells under appropriate conditions. We propose that trypsin treatment enhances the cytoaffinity of chickpea protein hydrolysates by exposing lysine and arginine residues that can interact with cell surface receptors. Our results suggest that chickpea protein hydrolysate functionalized microcarrier is a promising substrate for cultured meat production with cost-effectiveness and scalability.

Optimal temperature for culturing chicken satellite cells to enhance production yield and umami intensity of cultured meat

To investigate the optimal temperature at which to culture chicken muscle satellite cells (MSCs), to improve the production yield and umami intensity of cultured meat, chicken MSCs were cultured at 37, 39, 41, and 43 °C. The most active proliferation and greatest differentiation of MSCs was observed at 41 °C. A significantly shorter doubling time of MSCs and higher area percentage of differentiated myotubes and myofibers were observed at a culture temperature of 41 °C (p < 0.05). Consequently, the total amino acid content and free glutamic acid in cultured muscle tissue (CMT) at 41 °C was significantly higher than at other temperatures (p < 0.05). In addition, the umami and richness intensity of CMT was significantly higher at 41 °C than at other culture temperatures (p < 0.05). Results suggest that chicken MSCs proliferated more actively and differentiated optimally at 41 °C, resulting in a higher amino acid content and umami intensity of CMT.

Optimal Pre-Plating Method of Chicken Satellite Cells for Cultured Meat Production.

To establish a pre-plating method of chicken satellite cells with high purity, pre-plating was performed under culture conditions of 37°C and 41°C, and the pre-plating time was set from a total of 3 hours to 6 hours in consideration of the cell attachment time. The purity of the cells was confirmed by staining paired box protein 7 (Pax7) after proliferation, and Pax7 expression was the highest in culture flasks shaken for 2 hours after incubation at 41°C for 2 hours to prevent the attachment of satellite cells (p<0.05). Also, when pre-plating and proliferation were performed at 37°C and 41°C, the Pax7 expression rate was higher at 41°C. The differentiation capabilities of the three groups (T3, T6, and T7) with high Pax7 expression were compared and the fusion index (%) and myotube formation area (%) determined by myosin heavy chain (MHC) staining was calculated. The T6 and T7 groups, which were cultured at 41°C, showed significantly higher values than the T3 group (p<0.05). There was no significant difference in the expression of Pax7 and MHC between the T6 and T7 groups (p>0.05). These results suggest that pre-plating at 41°C for a total of 4 hours was the most efficient in terms of cost and time for purifying chicken satellite cells for cultured meat.

Effect of Different Basal Culture Media and Sera Type Combinations on Primary Broiler Chicken Muscle Satellite Cell Heterogeneity during Proliferation and Differentiation.

Simple SummaryLittle consistency in the literature exists for optimal culture conditions for proliferating and differentiating primary broiler chicken muscle satellite cells regarding basal culture media, proliferation sera, and differentiation sera. This experiment assessed primary satellite cell proliferation and differentiation when cultured in different combinations of basal media and sera. Cells were cultured in different basal media: low glucose Dulbecco’s Modified Eagle’s medium, McCoy’s 5A, and high glucose Dulbecco’s Modified Eagle’s medium. Each media was supplemented with 15% chicken serum, or a combination of 5% horse serum + 10% chicken serum during proliferation while 3% horse serum or 3% chicken serum were supplemented during differentiation. Cultures were immunofluorescence stained for myogenic regulatory factors at different time points during proliferation and differentiation. During proliferation and differentiation, cells cultured in Dulbecco’s Modified Eagle’s medium tended to have higher proportions of myogenic cells expressing myogenic regulatory factors and promoted satellite cell fusion into myotubes compared with McCoy’s 5A. Low glucose media, glucose concentration similar to circulating glucose concentrations in broilers, combined with sera published in the literature may be the optimal culture media to promote satellite cell proliferation and differentiation.The objective of this experiment was to access primary satellite cell (SC) proliferation and differentiation when cultured in different combinations of basal media and sera due to little consistency being published on the optimal culture media for primary broiler chicken satellite cells. Cells were cultured in one of three different basal media: McCoy’s 5A, high glucose Dulbecco’s Modified Eagle’s medium (DMEM), and low glucose DMEM. Media were supplemented with 15% chicken serum (CS) or a combination of 5% horse serum (HS) + 10% CS during proliferation while 3% HS or 3% CS were added to the media during differentiation. Cultures were immunofluorescence stained for myogenic regulatory factors (MRF) at 48, 72, and 96 h post-plating for proliferation (Pax7, MyoD, and Myf-5) and 96 h post-proliferation during differentiation (Pax7 and MyoD), including MF20 to assess fusion. Cells cultured in Dulbecco’s Modified Eagle’s medium tended to have higher proportions of myogenic cells expressing MRF during proliferation and promoted fusion into myotubes compared with McCoy’s 5A during differentiation. Culturing primary SC in low glucose media, glucose concentrations similar to circulating glucose concentrations in broilers, HSCS during proliferation and CS during differentiation, appears to be optimal for promoting broiler chicken satellite cell proliferation and differentiation.

Effects of 20(S)-hydroxycholesterol on satellite cell proliferation and differentiation of broilers

In the modern poultry industry, with increasing product demand, muscle growth rate and meat yield in chickens have tremendously changed. Understanding the regulation of muscle development is important to maintain efficient growth and development in meat-type chickens. 20(S)-hydroxycholesterol (20S) is known as one of the naturally occurring osteogenic cholesterol derivatives due to its ability to induce osteogenic differentiation; however, no studies have evaluated myogenic response to 20S in chicken muscle cells. To determine the use of 20S in vitro for the proliferation and differentiation of chicken satellite cells, satellite cells were isolated from pectoralis major muscle of 4-week-old Ross 708 male chickens and subjected to 0.25, 0.5, and 1.0 μmol of 20S during their proliferation and differentiation stages. Cell proliferation and differentiation were measured every 24 h for 72 h by determining DNA concentration, the activity of creatine kinase, and the expressions of myogenic regulatory transcription factors. Together these results suggested that a lower concentration of 20S did not affect myogenesis but a high concentration of 1.0 μmol 20S can negatively affect proliferation and differentiation in chicken satellite cells.

Modulatory effect of heat stress on viability of primary cultured chicken satellite cells and expression of heat shock proteins ex vivo

Satellite cells promote muscle repairing and muscle growth. Thereby the intention of the present study was to investigate the beneficial effects of heat stress at different time intervals on chicken satellite cells’ viability. Satellite cells were isolated from 1-day-old chicks and treated at two different temperatures (37 °C and 41 °C) for various time periods (6 h, 12 h, 24 h, 48 h, and 72 h). Both temperatures significantly increased cell viability after 24 h and 48 h. After 12 h, cell viability was significantly increased at 41 °C compared to 37 °C. However, more apoptotic cells were observed at end of the experiment of 41 °C compared to 37 °C. In addition, more live cells were found at early of experimental period at 41 °C than 37 °C. Additionally, protein and mRNA expression of HSP70, HP60 and HSP47 were significantly upregulated throughout the experimental period at temperature of 41 °C compared to those at 37 °C. These results indicate that cell viability and expression of heat stress related proteins/genes are induced by high temperature of 41 °C via heat stress pathway whereas activation of heat stress related proteins/genes are lower at 37 °C. Thus, 41 °C can trigger satellite cells’ viability essential for better cell survival than 37 °C at early incubation time.

KLF5 regulates chicken skeletal muscle atrophy via the canonical Wnt/β-catenin signaling pathway.

Recent studies in mice suggested that KLF5 (Kruppel like factor 5), a zinc-finger transcription factor, plays an important role in skeletal muscle development and regeneration. As an important factor in the process of muscle development, KLF5 participates in the regulation of the cell cycle, cell survival, and cell dryness under different environmental conditions, but it is not clear whether KLF5 participates in muscle atrophy. Therefore, we investigated whether KLF5 can regulate the atrophy of chicken satellite cells in vitro and examined its mechanism of action. qPCR showed that KLF5 gene knockdown promoted the expression of key genes in muscle atrophy. Subsequently, we sequenced and analyzed the transcriptomes of KLF5 silenced and control cells, and we showed that the differentially expressed genes were mainly enriched in 10 signaling pathways (P<0.05), with differential gene and enrichment analyses indicating that the Wnt signaling pathways are extremely important. In conclusion, our results indicate that KLF5 may regulate the atrophy of chicken skeletal muscle through the Wnt/β-catenin signaling pathway.

KLF5 functions in proliferation, differentiation, and apoptosis of chicken satellite cells.

KLF5 is an important regulator of cell proliferation, differentiation, and apoptosis in mammals. Little is known about the function of KLF5 in the regulation of chicken. Hence, qPCR was used to detect the expression of KLF5 in different tissues of chicken. And chicken skeletal muscle satellite cells (SMSCs) were transfected KLF5-specific small interfering RNA (siRNA) to assay SMSCs' proliferation, differentiation, and apoptosis. The results showed that KLF5 expressed higher in skeletal muscle than in the other tissues of chicken. Knockdown of KLF5 significantly inhibited the differentiation and increased apoptosis of chicken SMSCs, but it had no significant effect on proliferation of SMSCs. These results indicate that KLF5 plays an essential role during myogenesis, which will affect muscle repair and muscle regeneration, and may ameliorate muscle aging or sarcopenia.

FHL3 negatively regulates the differentiation of skeletal muscle satellite cells in chicken.

As a member of four and a half LIM domain proteins, FHL3 gene-encoded protein (FHL3) plays an important role in skeletal muscle development and contraction. In this study, we determined the potential role of FHL3 in the proliferation and differentiation of primary satellite cells in chicken. RT-qPCR results showed that FHL3 mRNA was highly expressed in skeletal muscle in 12 chicken tissues. Four cell proliferation assays (CCK8 assay; EDU staining assay; flow cytometric detection of cell cycle assay; and detection of cell proliferation marker genes Ki67 and N-Ras assay) revealed that FHL3 knockdown had no effect on the proliferation rate of chicken satellite cells. FHL3 knockdown promoted the differentiation of satellite cells into myotubes, as evidenced by increased fusion index, number of nuclei per myotube, Myog, Myh7, Myf5, and Mrf4 mRNA expressions, and myog and myosin heavy chain protein expressions of myogenic markers (P < 0.05). These results showed that the FHL3 was a negative regulator of the differentiation and fusion of chicken satellite cells into myotubes. However, FHL3 expression was increased during the differentiation of chicken satellite cells into myotubes. The study suggested that FHL3 might have different functions in chicken myotubes compared with that in chicken satellite cells.

Knockdown of CSRP3 inhibits differentiation of chicken satellite cells by promoting TGF-β/Smad3 signaling

Muscle LIM protein (MLP/CSRP3/CRP3) is a microtubule-associated protein preferentially expressed in cardiac and skeletal muscle and has a central role during muscle development and for architectural maintenance of muscle cells. LIM-domain proteins act as both modulators and downstream targets of TGF-β signaling, which is well documented to negatively regulate differentiation of myogenic precursor cells or myoblasts. Herein, we determined whether CSRP3 regulates chicken satellite cell proliferation and differentiation in vitro, and examined its mechanism of action by focusing on the TGF-β signaling pathway. Interference of CSRP3 mRNA expression had no effect on the proliferation of satellite cells, but significantly inhibited satellite cell differentiation into myotubes at 24, 48, and 72 h after initiation of differentiation. However, CSRP3 overexpression did not affect the proliferation or differentiation of satellite cells. Moreover, knockdown of CSRP3 caused up-regulation of TGF-β and Smad3 mRNA and protein levels. The phosphorylation of Smad3 in CSRP3-knockdown cells was greater than that in wild-type cells at 24, 48, and 72 h after initiation of differentiation. Collectively, knockdown of CSRP3 suppressed chicken satellite cell differentiation by regulating Smad3 phosphorylation in the TGF-β signaling pathway. Our results indicate that CSRP3 might play an important role in promoting satellite cell differentiation in chicken.

Effect of incubation temperature on neuropeptide Y and neuropeptide Y receptors in turkey and chicken satellite cells

Neuropeptide Y (NPY) is an appetite stimulating peptide released from the central nervous system and impacts the function of many different cell types. A recent transcriptome study showed that NPY expression was altered when turkey breast muscle satellite cells were incubated at low or high temperatures, suggesting NPY may mediate temperature effects on satellite cells. However, to date minimal information exists describing the expression and function of NPY in satellite cells. The objective of this study was to determine how temperature impacts NPY and NPY receptor gene expression in satellite cells isolated from turkeys and chickens with differing genetic lineages. Two broiler and two turkey breast muscle satellite cell lines were incubated at 35, 38 or 41 °C during proliferation and differentiation. In both turkey lines, NPY, and receptors NPY2R and NPY5R expression increased at elevated temperatures after 72 h of proliferation. During differentiation NPY and NPY5R expression increased in both turkey lines with higher temperatures, whereas NPY2R was minimally affected by temperature. In contrast, in both chicken cell lines there were few significant differences for NPY and NPY receptor expression across temperature during proliferation. During differentiation, the temperature effect was different in the two chicken cell lines. In the BPM8 chicken line, there were few differences in NPY and NPY receptors across temperature; whereas elevated temperatures increased NPY, NPY2R, and NPY5R expression in the 708 line. The differences between turkey and chicken lines suggest NPY has species specific satellite cell functions in response to heat stress.

Beta-carotene preferentially regulates chicken myoblast proliferation withdrawal and differentiation commitment via BCO1 activity and retinoic acid production

The enzyme β-carotene oxygenase 1 (BCO1) catalyzes the breakdown of provitamin A, including beta-carotene (BC), into retinal, prior to its oxidation into retinoic acid (RA). Allelic variation at the BCO1 locus results in differential expression of its mRNA and affects carotenoid metabolism specifically in chicken Pectoralis major muscle. In this context, the aim of this study was to evaluate the potential myogenic effect of BC and the underlying mechanisms in chicken myoblasts. BCO1 mRNA was detected in myoblasts derived from chicken satellite cells. Treating these myoblasts with BC led to a significant decrease in BrdU incorporation. This anti-proliferative effect was confirmed by a cell cycle study using flow cytometry. BC also significantly increased the differentiation index, suggesting a positive effect on the commitment of avian myoblasts to myogenic differentiation. Addition of DEAB, a specific inhibitor of RALDH activity, significantly reduced BC anti-proliferative and pro-differentiating effects, suggesting that BC exerted its biological effect on chicken myoblasts through activation of the RA pathway. We also observed that in myoblast showing decreased BCO1 expression consecutive to a natural mutation or to a siRNA treatment, the response to BC was inhibited. Nevertheless, BCO1 siRNA transfection increased expression of BCO2 which inhibited cell proliferation in control and BC treated cells.

Function of death-associated protein 1 in proliferation, differentiation, and apoptosis of chicken satellite cells

Muscle growth and regeneration are processes closely associated with proliferation, differentiation, and apoptosis of muscle cells. Death-associated protein 1 (DAP1) has been identified as a negative regulator of autophagy. Little is known about the function of DAP1 in the regulation of myogenesis and satellite cells. Chicken satellite cells were transfected with DAP1 cloned into the pCMS-enhanced green fluorescent protein vector or pcDNA3.1 vector, or a small interference RNA against the endogenous DAP1 gene. The cells were assayed for proliferation, differentiation, and apoptosis. The overexpression of DAP1 increased proliferation, differentiation, and myotube diameter, but it had no effect on satellite cell apoptosis. In contrast, knockdown of DAP1 significantly decreased proliferation, differentiation, and number of nuclei per myotube, and it increased apoptosis of the cells. DAP1 is required for regulating myogenesis and apoptosis of satellite cells, which may affect muscle mass accretion and regeneration, and ameliorate muscle sarcopenia.

Effect of lipids on avian satellite cell proliferation, differentiation and heparan sulfate proteoglycan expression

The objective of this study was to determine the effects of fatty acids on the proliferation, differentiation, and expression of syndecan-4 and glypican-1 in avian myogenic satellite cells (SC). SC derived from the pectoralis major (PM) and biceps femoris (BF) muscles of the turkey and chicken were individually administered 8 different fatty acids in defined medium during proliferation. A parallel set of turkey SC was induced to differentiate. Highest levels of proliferation of turkey PM and BF SC occurred in cultures containing oleate. Linoleate and oleate were equipotent in supporting proliferation of chicken SC. Microscopic examination revealed that inclusion of docosahexaenoate or eicosapentaenoate was toxic towards both PM and BF SC from both species. Linolenate and arachidonate diminished levels of differentiation. Expression of glypican-1 varied between treatments to a greater extent with turkey BF than with PM SC. Expression in chicken PM and BF SC demonstrated a similar pattern in response to treatments. Turkey PM syndecan-4 expression varied between treatments, whereas expression in turkey BF SC was similar between treatments. Expression in chicken SC varied little between treatments. The results demonstrate species and muscle-specific differences in the parameters examined. It is proposed that changes in lipid raft receptor interactions may contribute to these observed differences.

The role of myostatin in chicken ( Gallus domesticus) myogenic satellite cell proliferation and differentiation

Myostatin (GDF-8) inhibits the activation, proliferation, and differentiation of myogenic satellite cells. The relative importance of this growth factor is demonstrated in myostatin-null mice and cattle possessing defective myostatin genes. These defects result in greatly enhanced musculature. In the present study, the effect of myostatin on chicken myogenic satellite cells derived from two different skeletal muscles was studied. The effect of anti-myostatin antibodies on cellular responses was also examined. Satellite cells isolated from the pectoralis major (PM) muscles were more responsive to the proliferation depressing effects of myostatin compared to cells from the biceps femoris (BF; P ⩽ 0.05). Myostatin inhibited differentiation of satellite cells derived from the PM muscle ( P ⩽ 0.05), but had no effect on cells from the BF ( P ⩾ 0.05). Myostatin administered to proliferating cells depressed the synthesis of decorin ( P ⩽ 0.05), an extracellular matrix proteoglycan. However, in differentiating cultures, only BF cells expressed lower levels of decorin ( P ⩽ 0.05). Decorin expression in PM cells was unchanged ( P ⩾ 0.05). Administration of anti-myostatin antibodies to proliferating cultures increased cell proliferation by 6–7% over 3 days. There was no effect on differentiation of either PM or BF cells. The present study demonstrates that there are differences in the responsiveness to myostatin of chicken satellite cells derived from different muscles. Evidence is also given to support the role of endogenous myostatin in autocrine regulation of muscle growth.

Myotube morphology, and expression and distribution of collagen type I during normal and low score normal avian satellite cell myogenesis

Myogenic satellite cells are essential for postnatal muscle growth and the regeneration of muscle in response to injury. An understanding of how the extracellular matrix affects satellite cell activity, and the temporal and spatial expression of extracellular matrix macromolecules is largely unknown. In the avian genetic muscle weakness, low score normal (LSN), satellite cell proliferation and differentiation rates are significantly lower than that observed in normal chicken satellite cells, which may be attributed to a late embryonic increase in the expression of decorin. Satellite cell-derived morphological properties, collagen type I expression, and the spatial distribution of collagen type I were investigated during normal and LSN satellite cell proliferation and differentiation. These studies showed a decrease in LSN myotube length and the number of nuclei per myotube. Collagen type I expression was similar between the LSN and normal satellite cell cultures during the course of proliferation and differentiation. However, the spatial distribution of collagen type I was altered in the LSN cultures 48 h after the initiation of fusion. The LSN cultures exhibited a premature extracellular distribution of collagen type I compared to the normal satellite cells.

Regulation of amino acid transport and protein metabolism in myotubes derived from chicken muscle satellite cells by insulin‐like growth factor‐I

The effects of insulin and insulin-like growth factor-I (IGF-I) on amino acid transport and protein metabolism were compared in myotubes derived from chicken breast muscle satellite cells. Protein synthesis was assessed by continuous labelling with [3H]-tyrosine. Protein degradation was estimated by the release of trichloroacetic acid (TCA) soluble radioactivity by cells which had been previously labelled with [3H]-tyrosine for 3 days. Amino acid transport was measured in myotubes incubated in Dulbecco's modified Eagle's medium (DMEM) 0.5% bovine serum albumin (BSA) with or without insulin or IGF-I. Subsequent [3H]-aminoisobutyric acid (AIB) uptake was then measured in amino acid-free medium. IGF-I was more efficient than insulin at equimolar concentration (3.2 nmol/l) in stimulating protein synthesis (127 and 113% of basal, respectively) and inhibiting protein degradation (32% and 13% inhibition of protein degradation following 4 h incubation). Half maximal effective concentrations for stimulation of AIB uptake were 0.27 +/- 0.03 nmol/l and 34.8 +/- 3.1 nmol/l for IGF-I and insulin respectively, with maximal stimulation of about 340% of basal. Cycloheximide (3.6 mumol/l) diminished IGF-I-stimulated AIB uptake by 55%. Chicken growth hormone had no effect on basal AIB uptake in these cells and neither glucagon nor dexamethasone had an effect on basal or IGF-I-stimulated AIB uptake. This study demonstrates an anabolic effect for IGF-I in myotubes derived from primary chicken satellite cells which is mediated by the type I IGF receptor, since the cation-independent mannose 6-phosphate receptor does not bind IGF-II in chicken cells.

P53 Gene Is Up-Regulated during Skeletal Muscle Cell Differentiation

Differentiation of cells in the mouse myogenic cell line C2, or a primary culture of chicken satellite cells was induced by low serum levels in the growth medium. Endogenous wild-type p53 mRNA was substantially expressed after approximately 5 h of incubation. Induction of p53 mRNA expression was also observed in cells treated with 10 −8 M retinoic acid, after 18 h of incubation. In both cases, the increase in p53 mRNA was transient. c-fos mRNA levels decreased rapidly and were barely detectable after 2 h of exposure to retinoic acid. The down-regulation of c-fos confirms its role in muscle cell differentiation, whereas the up-regulation of wild-type p53 suggests its role during this process.

Retinoic acid induces adult muscle cell differentiation mediated by the retinoic acid receptor‐α

Retinoic acid (RA) is known to induce differentiation in many cell systems. This induction is mediated by nuclear RA receptors (RARs), which act as transactivating factors belonging to the superfamily of steroid/thyroid nuclear receptors. In this study, effects of RA were studied in a mouse myogenic C2 cell line and in primary chicken satellite cells, the myogenic precursor cells in adult muscle. Addition of RA decreased the rate of DNA synthesis in a dose-dependent manner in both cultures. Differentiation was more rapid in cells treated with RA, and these cells exhibited large myotubes after 48 hr of incubation as compared to untreated cells, which were still proliferating. Addition of 10(-8) M RA to C2 cells elevated mRNA levels of myogenin, a skeletal muscle-specific transcription factor. In parallel, activity levels of the muscle-specific creatine kinase were enhanced in the RA-treated cells nearly twofold as compared to the untreated cells. RA treatment of both mouse C2 and chicken satellite cells caused rapid induction of the RAR-alpha mRNA levels. Maximal mRNA levels were observed after 2 to 5 hr followed by a sharp reduction to nearly zero levels at 9 hr. The RAR-alpha mRNA levels augmented in a dose-dependent manner between concentrations of 10(-10) M and 10(-8) M RA, whereas higher concentrations caused mRNA levels to decrease. These results indicate that RA induces differentiation in both adult skeletal muscle primary satellite cells and a myogenic cell line. The rapid and specific induction of RAR-alpha mRNA in these cells upon exposure to RA may suggest that this receptor is the primary target and a mediator of RA.