Satellite Cell Population Research Articles

Isolation of a persistently quiescent muscle satellite cell population.

Although studies have identified characteristics of quiescent satellite cells (SCs), their isolation has been hampered by the fact that the isolation procedures result in the activation of these cells into their rapidly proliferating progeny (myoblasts). Thus, the use of myoblasts for therapeutic (regenerative medicine) or industrial applications (cellular agriculture) has been impeded by the limited proliferative and differentiative capacity of these myogenic progenitors. Here we identify a subpopulation of satellite cells isolated from mouse skeletal muscle using flow cytometry that is highly Pax7-positive, exhibit a very slow proliferation rate (7.7 ± 1.2 days/doubling), and are capable of being maintained in culture for at least 3 mo without a change in phenotype. These cells can be activated from quiescence using a p38 inhibitor or by exposure to freeze-thaw cycles. Once activated, these cells proliferate rapidly (22.7 ± 0.2 h/doubling), have reduced Pax7 expression (threefold decrease in Pax7 fluorescence vs. quiescence), and differentiate into myotubes with a high efficiency. Furthermore, these cells withstand freeze-thawing readily without a significant loss of viability (83.1 ± 2.1% live). The results presented here provide researchers with a method to isolate quiescent satellite cells, allowing for more detailed examinations of the factors affecting satellite cell quiescence/activation and providing a cell source that has a unique potential in the regenerative medicine and cellular agriculture fields.NEW & NOTEWORTHY We provide a method to isolate quiescent satellite cells from skeletal muscle. These cells are highly Pax7-positive, exhibit a very slow proliferation rate, and are capable of being maintained in culture for months without a change in phenotype. The use of these cells by muscle researchers will allow for more detailed examinations of the factors affecting satellite cell quiescence/activation and provide a novel cell source for the regenerative medicine and cellular agriculture fields.

Satellite cells in the growth and maintenance of muscle.

Embryonic skeletal muscle growth is contingent upon a population of somite derived satellite cells, however, the contribution of these cells to early postnatal skeletal muscle growth remains relatively high. As prepubertal postnatal development proceeds, the activity and contribution of satellite cells to skeletal muscle growth diminishes. Eventually, at around puberty, a population of satellite cells escapes terminal commitment, continues to express the paired box transcription factor Pax7, and reside in a quiescent state orbiting the myofiber periphery adjacent to the basal lamina. After adolescence, some satellite cell contributions to muscle maintenance and adaptation occur, however, their necessity is reduced relative to embryonic, early postnatal, and prepubertal growth.

CRIPTO-based micro-heterogeneity of mouse muscle satellite cells enables adaptive response to regenerative microenvironment

Skeletal muscle repair relies on heterogeneous populations of satellite cells (SCs). The mechanisms that regulate SC homeostasis and state transition during activation are currently unknown. Here, we investigated the emerging role of non-genetic micro-heterogeneity, i.e., intrinsic cell-to-cell variability of a population, in this process. We demonstrate that micro-heterogeneity of the membrane protein CRIPTO in mouse-activated SCs (ASCs) identifies metastable cell states that allow a rapid response of the population to environmental changes. Mechanistically, CRIPTO micro-heterogeneity is generated and maintained through a process of intracellular trafficking coupled with active shedding of CRIPTO from the plasma membrane. Irreversible perturbation of CRIPTO micro-heterogeneity affects the balance of proliferation, self-renewal, and myogenic commitment in ASCs, resulting in increased self-renewal invivo. Our findings demonstrate that CRIPTO micro-heterogeneity regulates the adaptative response of ASCs to microenvironmental changes, providinginsights into the role of intrinsic heterogeneity in preserving stem cell population diversity during tissue repair.

Skeletal muscle regeneration failure in ischemic-damaged limbs is associated with pro-inflammatory macrophages and premature differentiation of satellite cells

BackgroundChronic limb-threatening ischemia (CLTI), a severe manifestation of peripheral arterial disease (PAD), is associated with a 1-year limb amputation rate of approximately 15–20% and substantial mortality. A key feature of CLTI is the compromised regenerative ability of skeletal muscle; however, the mechanisms responsible for this impairment are not yet fully understood. In this study, we aim to delineate pathological changes at both the cellular and transcriptomic levels, as well as in cell–cell signaling pathways, associated with compromised muscle regeneration in limb ischemia in both human tissue samples and murine models of CLTI.MethodsWe performed single-cell transcriptome analysis of ischemic and non-ischemic muscle from the same CLTI patients and from a murine model of CLTI. In both datasets, we analyzed gene expression changes in macrophage and muscle satellite cell (MuSC) populations as well as differential cell–cell signaling interactions and differentiation trajectories.ResultsSingle-cell transcriptomic profiling and immunofluorescence analysis of CLTI patient skeletal muscle demonstrated that ischemic-damaged tissue displays a pro-inflammatory macrophage signature. Comparable results were observed in a murine CLTI model. Moreover, integrated analyses of both human and murine datasets revealed premature differentiation of MuSCs to be a key feature of failed muscle regeneration in the ischemic limb. Furthermore, in silico inferences of intercellular communication and in vitro assays highlight the importance of macrophage-MuSC signaling in ischemia induced muscle injuries.ConclusionsCollectively, our research provides the first single-cell transcriptome atlases of skeletal muscle from CLTI patients and a murine CLTI model, emphasizing the crucial role of macrophages and inflammation in regulating muscle regeneration in CLTI through interactions with MuSCs.

236 Assessment of Early-Stage Thermal Manipulation on Broiler Chicken Muscle Satellite Cell Populations at Transfer and Hatch

Abstract Multi-stage hatching egg incubators are challenging to manage but still common throughout the US broiler industry. It is difficult to satisfy all embryonic temperature requirements in systems where late-stage embryos must be kept from overheating while ensuring early-stage embryos are not at sub-optimal temperatures. Our previous work showed improvements in performance and meat yield when incubation temperature was increased to 38.6 °C from embryonic day (ED) 4 to 11 as opposed to lowering it to 36.4 °C compared with a 37.5 °C control. The objective of this work was to assess the effect of thermal variation (TV) during early-stage incubation (ESI) on muscle satellite cell (SC) heterogeneity in broiler chicks at transfer and hatch to explore the mechanism responsible for increased meat yield in our previous study. Broiler breeder eggs (n = 2,160) were incubated at 37.5 °C from embryonic day (ED) 0 to 3. On ED 4, COLD incubator set points decreased to 36.4 °C, HOT incubator set points increased to 38.6 °C, and control incubators remained 37.5 °C (n = 2 incubators per treatment). On ED 11, all incubators were set to 37.5 °C until ED 18 when eggs were transferred to hatchers. At transfer (ED 18) and hatch (ED 21), pectoralis major (PM) and biceps femoris (BF) muscle samples were collected from 6 chicks per treatment. Samples were immunofluorescence stained to facilitate taxonomy of SC populations expressing the myogenic regulatory factors and SC markers, MyoD, MRF4, and Myf5, by fluorescence microscopy. Data were analyzed as a 1-way ANOVA with the GLIMMIX procedure of SAS. Means were separated at P ≤ 0.05 with the PDIFF option. Tendencies were declared when 0.0501 ≤ P ≤ 0.10. Chicks from COLD incubators had the greatest density of MyoD+:Myf5+ SC in their BF (P = 0.0261) and tended to have a greater density of MyoD+:MRF4+ SC in their PM muscle (P = 0.0562) compared with chicks from HOT incubators. On ED 21, chicks from HOT incubators had the greatest density of MyoD+:MRF4+:Myf5+ SC in their PM muscle (P = 0.0406). Temperature gradients as small as 1.1 °C during ESI altered BF SC populations in chicks at transfer and PM SC populations at both transfer and hatch. Overall, these results indicate that TV during ESI can influence SC population kinetics in both PM and BF muscles of modern, fast-growing, high-yielding broiler chicks at ED 18 and 21. Further investigation is needed to understand the longer-term effects on SC populations and the cell signaling pathways involved over time during the post-hatch grow out period to explain the meat yield responses observed in our previous work.

270 Cellular Response to Heat Exposure: Influences on the Growth Potential of Bovine Skeletal Muscle and Adipose Tissue

Abstract Our lack of understanding regarding how heat exposure affects major tissue growth and meat attributes impedes our ability to counteract more frequent climate extremes and promote profitability in the beef cattle industry. The objective of the study was to determine pathways that heat shock proteins (HSPs) regulate in beef cattle’s skeletal muscle and adipose tissue growth and metabolism. Bovine satellite cells (BSCs) were isolated from the longissimus muscle of 3-month-old Holstein bulls (n = 5). Preadipose cells were isolated from the subcutaneous fat of 18-month-old Angus steers (n = 5) and induced adipogenic differentiation. Satellite, myofibers, and adipose cells were incubated at 38°C (control; CON), 39.5°C (moderate heat stress; MHS), and 41°C (extreme heat stress; EHS) and harvested for mRNA gene expressions, protein level, proteomics, immunostaining. Heat exposure cued bovine satellite cell proliferation and may increase the self-renewing satellite cell population. After 3 h of heat exposure, the mRNA gene expression and protein levels of Pax3 and Myf5 were downregulated, while HSPs 20, 27, 70, and 90 were upregulated. Small heat shock proteins, especially HSP27, appear to be essential in regulating satellite cell activity under heat stress. When HSP27 knock-down BSCs were exposed to EHS for 3 h, Myf5 gene expressions and protein levels returned to normal. This may indicate that HSP27 directly regulates Myf5 under heat stress and temporarily activates the self-renewal mechanism instead of committing myogenic differentiation. Results from proteomics showed that pathways related to oxidative phosphorylation, NADH dehydrogenase activity, and transmembrane transport were upregulated by EHS while MHS upregulated biosynthesis of amino acids, PI3K-Akt signaling pathway. Heat exposure during myogenic differentiation of satellite cells seemingly stimulates the commitment of myogenesis. Heat treatments increased mRNA gene expressions of IGF I, MHC I, MyoD, and myogenin and protein levels. Heat exposure also improved the muscle protein synthesis pathway by regulating the phosphorylation of mTOR, Akt, and P70S6. Heat stress triggered adipogenic differentiation and lipid storage, followed by increased expression of HSP. Extreme heat stress increased adipose cell growth by upregulating C/EBPα and PPARγ, key regulators of adipogenesis. Lipogenic genes FAS and SCD were also increased, accompanied by HSPs. Adipocytes exposed to EHS also increased intracellular lipid accumulation. Proteomic analysis revealed that carbon and pyruvate metabolism pathways were upregulated by MHS, whereas glycolysis/ gluconeogenesis, protein processing in the endoplasmic reticulum, RNA transport, and biosynthesis of amino acids were upregulated by EHS. Cellular mechanisms related to skeletal muscle hypertrophy and adipose tissue growth are responsive in in-vitro settings; however, the results are controversial depending on cell type and animal species. Various factors such as metabolic, hormonal, and immunologic regulation need to be considered to accurately assess the effect of HS on the postnatal growth of major tissues at the animal level.

Identification of transition factors in myotube formation from proteome and transcriptome analyses

Muscle fibers are the main component of skeletal muscle and undergo maturation through the formation of myotubes. During early development, a population of skeletal muscle satellite cells (SSCs) proliferate into myoblasts. The myoblasts then undergo further differentiation and fusion events, leading to the development of myotubes. However, the mechanisms involved in the transition from SSCs to myotube formation remain unclear. In this study, we characterized changes in the proteomic and transcriptomic expression profiles of SSCs, myoblasts (differentiation for 2 d) and myotubes (differentiation for 10 d). Proteomic analysis identified SLMAP and STOM as potentially associated with myotube formation. In addition, some different changes in MyoD, MyoG, Myosin7 and Desmin occurred after silencing SLMAP and STOM, suggesting that they may affect changes in the myogenic marker. GO analysis indicated that the differentiation and migration factors SVIL, ENSCHIG00000026624 (AQP1) and SERPINE1 enhanced the transition from SSCs to myoblasts, accompanied by changes in the apoptotic balance. In the myoblast vs. myotube group, candidates related to cell adhesion and signal transduction were highly expressed in the myotubes. Additionally, CCN2, TGFB1, MYL2 and MYL4 were identified as hub-candidates in this group. These data enhance our existing understanding of myotube formation during early development and repair.

Broiler breast muscle myopathies: association with satellite cells

Heavy weight fast-growing meat-type broiler chickens have largely been selected for growth rate, muscle mass yield especially for the breast muscle, and feed conversion. Substantial improvements have been made, but in recent years breast meat quality issues resulting in product downgrades or condemnation have occurred especially from necrotic and fibrotic myopathies like Wooden Breast. In general, the morphological structure of the broiler breast muscle has changed in the modern commercial broiler with muscle fiber diameters increased, circulatory supply decreased, and connective spacing between individual fibers and fiber bundles decreased. Satellite cells are the primary cell type responsible for all posthatch muscle growth, and the repair and regeneration of muscle fibers. Recent evidence is suggestive of changes in the broiler satellite cell populations which will limit the ability of the satellite cells to regenerate damaged muscle fibers back to their original. These changes in the cellular biology of broiler satellite cells are likely associated with the necrosis and fibrosis observed in myopathies like Wooden Breast.

Influence of Media Composition on the Level of Bovine Satellite Cell Proliferation.

It is predicted that already in 2040, 35% of requirements for meat will be provided by in vitro production. Recreating the course of myogenesis in vitro, and thus resembling a structure of muscle tissue, is the basis for research focusing on obtaining cultured meat and requires providing relevant factors supporting the proliferation of satellite cells-being precursors of skeletal muscles. The present work aimed to develop the composition of the medium that would most effectively stimulate the proliferation of bovine satellite cells (BSCs). The modeling and optimization methods included the measurements of the synergistic, co-stimulatory effect of three medium components: the amount of glucose, the type of serum (bovine or horse), and the amount of mitogenic factor-bFGF. Additionally, the qPCR analyses determined the expression of genes involved in myogenesis, such as Pax7 and Myogenic Regulatory Factors, depending on the level of the tested factor. The results showed significant positive effects of serum type (bovine serum) and mitogenic factor (addition of 10 ng/mL bFGF) on the proliferation rate. In turn, qPCR analysis displayed no significant differences in the relative expression level of Pax7 genes and MRF factors for both factors. However, a statistically higher Pax7 and Myf5 gene expression level was revealed when a low glucose medium was used (p < 0.05). In conclusion, the components of the medium, such as bovine serum and the addition of a mitogenic factor at the level of 10 ng/mL, ensure a higher proliferation rate of BSCs and lower glucose content ensured the expression of crucial genes in the self-renewal of the satellite cell population.

NEDD4-1 deficiency impairs satellite cell function during skeletal muscle regeneration

BackgroundSatellite cells are tissue-specific stem cells primarily responsible for the regenerative capacity of skeletal muscle. Satellite cell function and maintenance are regulated by extrinsic and intrinsic mechanisms, including the ubiquitin–proteasome system, which is key for maintaining protein homeostasis. In this context, it has been shown that ubiquitin-ligase NEDD4-1 targets the transcription factor PAX7 for proteasome-dependent degradation, promoting muscle differentiation in vitro. Nonetheless, whether NEDD4-1 is required for satellite cell function in regenerating muscle remains to be determined.ResultsUsing conditional gene ablation, we show that NEDD4-1 loss, specifically in the satellite cell population, impairs muscle regeneration resulting in a significant reduction of whole-muscle size. At the cellular level, NEDD4-1-null muscle progenitors exhibit a significant decrease in the ability to proliferate and differentiate, contributing to the formation of myofibers with reduced diameter.ConclusionsThese results indicate that NEDD4-1 expression is critical for proper muscle regeneration in vivo and suggest that it may control satellite cell function at multiple levels.

40 Texel Genetics Increase Muscle Growth by Altering Satellite Cell Characteristics

Abstract The Texel breed is a double muscled breed of sheep that is commonly used as a terminal sire in meat-production. The increase in muscle mass is related to a single nucleotide polymorphism in the myostatin gene, a myokine that inhibits muscle growth. There is limited information on how postnatal skeletal muscle fiber hypertrophy is altered in Texel lambs. Satellite cells or muscle stem cells have a major role in postnatal hypertrophy of muscle fibers. Satellite cells are generally found in a state of quiescence and must activate, proliferate, and differentiate to increase DNA content in myofibers during periods of postnatal muscle growth. The objective of this study was to evaluate muscle fiber hypertrophy and satellite cell proliferation and differentiation in lambs of two sire breeds, Texel or Suffolk. Male lambs of each sire breed (n = 4/sire breed/time) were harvested at d 2, 14, and 203 of age. Muscle samples were collected from the longissimus and snap frozen for examination of myofiber cross-sectional area by fluorescent immunohistochemistry. Another portion of the longissimus was used to isolate satellite cells for in vitro experiments to examine population number, proliferation, and differentiation capacity. Isolated satellite cells were counted, seeded for activation, and grown until colonies reached 80% confluence. Activated cells were seeded for a proliferation assay measuring DNA content daily for 6 days. Following proliferation, cells were differentiated for 4 days, and the number of differentiated nuclei were counted with fluorescent immunohistochemistry. Sire breed did not impact (P &amp;lt; 0.05) the weight of the longissimus; however, muscle ribeye area tended (P &amp;lt; 0.10) to be increased for Texel lambs compared with Suffolk lambs. In the early postnatal period, from d 2 to 14, longissimus mass increased (P &amp;lt; 0.05) by 160%, and ribeye area increased by 100%. Between d 4 and 203 longissimus mass increased (P &amp;lt; 0.05) by 350%, while longissimus area only increased by 100%. Muscle fiber cross sectional area expanded (P &amp;lt; 0.05) from d 14 to 203 of age, and cross-sectional area was greater (P &amp;lt; 0.05) for Texel than Suffolk lambs at d 203. Satellite cell populations per gram of tissue increased (P &amp;lt; 0.05) between d 2 and 14 and decreased from d 14 to 203. The number of satellite cells that activated from quiescence was reduced (P &amp;lt; 0.05) as animal age increased but was not impacted (P &amp;gt; 0.05) by sire breed. Following activation, satellite cell proliferation capacity was greater (P &amp;lt; 0.05) for Texel compared with Suffolk satellite cells. Differentiation capacity was not altered (P &amp;gt; 0.05) by sire breed. Texel-sired lambs had larger longissimus cross-sectional muscle fiber area at d 203 of age than Suffolk. These changes appear to be related to greater proliferation of satellite cells, the stem cells of muscle, to enhance muscle fiber hypertrophy during postnatal growth.

Single cell analysis reveals satellite cell heterogeneity for proinflammatory chemokine expression

Background: The expression of proinflammatory signals at the site of muscle injury are essential for efficient tissue repair and their dysregulation can lead to inflammatory myopathies. Macrophages, neutrophils, and fibroadipogenic progenitor cells residing in the muscle are significant sources of proinflammatory cytokines and chemokines. However, the inducibility of the myogenic satellite cell population and their contribution to proinflammatory signaling is less understood. Methods: Mouse satellite cells were isolated and exposed to lipopolysaccharide (LPS) to mimic sterile skeletal muscle injury and changes in the expression of proinflammatory genes was examined by RT-qPCR and single cell RNA sequencing. Expression patterns were validated in skeletal muscle injured with cardiotoxin by RT-qPCR and immunofluorescence. Results: Satellite cells in culture were able to express Tnfa, Ccl2, and Il6, within 2h of treatment with LPS. Single cell RNA-Seq revealed seven cell clusters representing the continuum from activation to differentiation. LPS treatment led to a heterogeneous pattern of induction of C-C and C-X-C chemokines (e.g., Ccl2, Ccl5, and Cxcl0) and cytokines (e.g., Tgfb1, Bmp2, Il18, and Il33) associated with innate immune cell recruitment and satellite cell proliferation. One cell cluster was enriched for expression of the antiviral interferon pathway genes under control conditions and LPS treatment. Activation of this pathway in satellite cells was also detectable at the site of cardiotoxin induced muscle injury. Conclusion: These data demonstrate that satellite cells respond to inflammatory signals and secrete chemokines and cytokines. Further, we identified a previously unrecognized subset of satellite cells that may act as sensors for muscle infection or injury using the antiviral interferon pathway.

Differential effects of temperature and mTOR and Wnt-planar cell polarity pathways on syndecan-4 and CD44 expression in growth-selected turkey satellite cell populations.

Satellite cells (SCs) comprise a heterogeneous population of muscle stem cells. Thermal stress during the first week after hatch alters proliferation, myogenesis, and adipogenesis of SCs of turkey pectoralis major (p. major) muscle via mechanistic target of rapamycin (mTOR) and wingless-type mouse mammary tumor virus integration site family/planar cell polarity (Wnt/PCP) pathways. Pivotal genes in mTOR and Wnt/PCP pathways are mTOR and frizzled-7 (Fzd7), respectively. The objective of this study was to determine the differential effects of thermal stress on SDC4 and CD44 expression in turkey p. major muscle SCs and how the expression of SDC4 and CD44 is modulated by the mTOR and Wnt/PCP pathways. Satellite cells were isolated from the p. major muscle of 1-week-old faster-growing modern-commercial (NC) turkeys and slower-growing historic Randombred Control Line 2 (RBC2) turkeys, and were challenged with hot (43°C) and cold (33°C) thermal stress for 72 h of proliferation followed by 48 h of differentiation. The NC line SCs were found to contain a lower proportion of SDC4 positive and CD44 negative (SDC4+CD44-) cells and a greater proportion of SDC4 negative and CD44 positive (SDC4-CD44+) cells compared to the RBC2 line at the control temperature (38°C) at both 72 h of proliferation and 48 h of differentiation. In general, at 72 h of proliferation, the proportion of SDC4+CD44- cells decreased with heat stress (43°C) and increased with cold stress (33°C) relative to the control temperature (38°C) in both lines, whereas the proportion of SDC4-CD44+ cells increased with heat stress and decreased with cold stress. In general, the expression of SDC4 and CD44 in the NC SCs showed greater response to both hot and cold thermal stress compared to the RBC2 cells. Knockdown of mTOR or Fzd7 expression increased the proportion of SDC4+CD44- cells while the proportion of SDC4-CD44+ cells decreased during differentiation with line differences being specific to treatment temperatures. Thus, differential composition of p. major muscle SCs in growth-selected commercial turkey may be resulted, in part, from the alteration in SDC4 and CD44 expression. Results indicate differential temperature sensitivity and mTOR and Wnt/PCP pathway responses of growth-selected SC populations and this may have long-lasting effect on muscle development and growth.

Differences in Inferior Oblique Muscle Satellite Cell Populations between Primary and Secondary Inferior Oblique Muscle Overaction

Introduction: The aim of the study was to compare the number of paired box 7 (PAX7)-positive, myogenic differentiation 1 (MyoD1)-positive, and neural cell adhesion molecule-1 (NCAM)-positive satellite cells in human primary versus secondary inferior oblique muscle overaction (IOOA). Methods: This prospective observational study enrolled patients who underwent inferior oblique muscle myectomy at the Department of Pediatric Ophthalmology and Strabismus in Tianjin Eye Hospital between January 2020 and November 2020. The muscle specimens were processed. Immunohistochemistry and immunofluorescence were used to quantify inferior oblique muscle fiber diameter and PAX7-positive, NCAM-positive, and MyoD1-positive satellite cells. Results: Thirty-eight patients with inferior oblique overaction were enrolled: 18 with primary IOOA and 20 with secondary IOOA. The participants were significantly younger in the secondary IOOA group than in the primary IOOA group (2.8 ± 1.2 vs. 9.0 ± 3.2 years, p < 0.001). The muscle fiber diameter between the two groups was not significantly different (13.5 ± 1.4 vs. 13.8 ± 0.7 µm, p = 0.530), but PAX7+ (3.3 ± 2.8 vs. 1.8 ± 0.6, p < 0.001), NCAM+ (3.6 ± 1.5 vs. 1.7 ± 0.2, p < 0.001), and MyoD1+ (4.8 ± 1.9 vs. 2.7 ± 0.5, p < 0.001) cell counts were higher in primary IOOA than in secondary IOOA. Conclusions: PAX7pos, MyoD-1pos, and NCAMpos cell counts per laminar nucleus were almost 2-fold higher in the primary IOOA group than in the secondary IOOA group.

Technical note: an optimized method to isolate, purify, and differentiate satellite cells from broiler chicks.

Development and maintenance of healthy muscle fibers rely on the myogenic potential of satellite cells (SC), muscle stem cells that proliferate and differentiate to form myotubes. Satellite cells are indispensable for post-hatch muscle growth as well as muscle repair and regeneration when myofibers are damaged. Pectoralis major of young broiler chicks (5-d olds) is a readily available source of SC, which can be used in vitro to elucidate cellular and molecular mechanisms responsible for muscle growth and regeneration in broilers. Here, we optimized a method for efficient isolation, purification, and differentiation of SC, from young broiler chicks. This procedure includes a simple method that allows SC to be purified from other muscle cell types that can impede the fidelity of follow-on experiments, particularly highly sensitive measures such as RNAseq. The methods for culturing and differentiating SC into multinucleated myotubes were also optimized by testing serum types, concentrations, and the effects of chicken embryo extract. Using the isolation procedure, a highly pure SC population (94.6 ± 2.11% Pax7+) with high viability and yield was obtained, and their capacity to differentiate into myotubes was confirmed. Enrichment for SC and myogenic capacity were maintained through multiple passages and after cryopreservation. Analysis of gene expression over the first 48 h of differentiation confirmed that SC exhibited the expected molecular signature of myogenesis. Taken together, this method simplifies the ability to isolate and maintain a relatively pure population of SC with strong myogenic potential from young broiler chicks, and should support downstream applications for assessing the impact of nutrients, metabolites, and other physiological cues on muscle growth and development in broilers.

MiR-106b is a novel target to promote muscle regeneration and restore satellite stem cell function in injured Duchenne dystrophic muscle

Satellite cells (SCs), muscle stem cells, display functional heterogeneity, and dramatic changes linked to their regenerative capabilities are associated with muscle-wasting diseases. SC behavior is related to endogenous expression of the myogenic transcription factor MYF5 and the propensity to enter into the cell cycle. Here, we report a role for miR-106b reinforcing MYF5 inhibition and blocking cell proliferation in a subset of highly quiescent SC population. miR-106b down-regulation occurs during SC activation and is required for proper muscle repair. In addition, miR-106b is increased in dystrophic mice, and intramuscular injection of antimiR in injured mdx mice enhances muscle regeneration promoting transcriptional changes involved in skeletal muscle differentiation. miR-106b inhibition promotes the engraftment of human muscle stem cells. Furthermore, miR-106b is also high in human dystrophic muscle stem cells and its inhibition improves intrinsic proliferative defects and increases their myogenic potential. This study demonstrates that miR-106b is an important modulator of SC quiescence, and that miR-106b may be a new target to develop therapeutic strategies to promote muscle regeneration improving the regenerative capabilities of injured dystrophic muscle.

Early-stage inflammation changes in supraspinatus muscle after rotator cuff tear.

Rotator cuff (RC) tendon tear leads to impaired shoulder function and pain. The supraspinatus (SS) tendon is most often affected, but the biological response of the SS muscle to SS tendon tear is largely unknown. This study aimed to investigate time-dependent muscle inflammation, degeneration, fatty infiltration, and regeneration in experimental SS tear conditions. Forty-five C57BL/6 mice were subjected to SS tendon tear and allowed to recover for 1, 3, 5, 7, 14, or 28 days. The extent of muscle damage was examined using histologic, flow cytometric, proteomic, and chemiluminescence analyses. We found that muscle inflammation peaked around day 5 with increased monocyte infiltration and increased cytokine levels in the ipsilateral compared to the contralateral SS muscle. Bioinformatics analysis of proteomics on mice that survived 5 days after RC tendon tear revealed upregulated proteins involved in "neutrophil activation involved in immune response" and "extracellular matrix organization," whereas "skeletal muscle tissue development and contraction" and "respiratory electron transport chain" were among the most downregulated. Histologic analysis of collagen showed increased collagen accumulation and fatty infiltration of the ipsilateral SS over time. Finally, we observed time- and lesion-dependent changes in satellite cell and fibro-adipogenic progenitor populations. Altogether, we demonstrate that the SS muscle shows severe signs of acute inflammation, early degeneration, and fatty infiltration, as well as reduced regenerative potential following SS tendon tear.

Myoblast deactivation within engineered human skeletal muscle creates a transcriptionally heterogeneous population of quiescent satellite-like cells

Satellite cells (SCs), the adult Pax7-expressing stem cells of skeletal muscle, are essential for muscle repair. However, in vitro investigations of SC function are challenging due to isolation-induced SC activation, loss of native quiescent state, and differentiation to myoblasts. In the present study, we optimized methods to deactivate in vitro expanded human myoblasts within a 3D culture environment of engineered human skeletal muscle tissues (“myobundles”). Immunostaining and gene expression analyses revealed that a fraction of myoblasts within myobundles adopted a quiescent phenotype (3D-SCs) characterized by increased Pax7 expression, cell cycle exit, and activation of Notch signaling. Similar to native SCs, 3D-SC quiescence is regulated by Notch and Wnt signaling while loss of quiescence and reactivation of 3D-SCs can be induced by growth factors including bFGF. Myobundle injury with a bee toxin, melittin, induces robust myofiber fragmentation, functional decline, and 3D-SC proliferation. By applying single cell RNA-sequencing (scRNA-seq), we discover the existence of two 3D-SC subpopulations (quiescent and activated), identify deactivation-associated gene signature using trajectory inference between 2D myoblasts and 3D-SCs, and characterize the transcriptomic changes within reactivated 3D-SCs in response to melittin-induced injury. These results demonstrate the ability of an in vitro engineered 3D human skeletal muscle environment to support the formation of a quiescent and heterogeneous SC population recapitulating several aspects of the native SC phenotype, and provide a platform for future studies of human muscle regeneration and disease-associated SC dysfunction.

Follistatin Protein Enhances Satellite Cell Counts in Reinnervated Muscle.

Background Muscle recovery following peripheral nerve repair is sup-optimal. Follistatin (FST), a potent muscle stimulant, enhances muscle size and satellite cell counts following reinnervation when administered as recombinant FST DNA via viral vectors. Local administration of recombinant FST protein, if effective, would be more clinically translatable but has yet to be investigated following muscle reinnervation. Objective The aim of this study is to assess the effect of direct delivery of recombinant FST protein on muscle recovery following muscle reinnervation. Materials and Methods In total, 72 Sprague-Dawley rats underwent temporary (3 or 6 months) denervation or sham denervation. After reinnervation, rats received FST protein (isoform FS-288) or sham treatment via a subcutaneous osmotic pump delivery system. Outcome measures included muscle force, muscle histomorphology, and FST protein quantification. Results Follistatin treatment resulted in smaller muscles after 3 months denervation ( p = 0.019) and reduced force after 3 months sham denervation ( p < 0.001). Conversely, after 6 months of denervation, FST treatment trended toward increased force output ( p = 0.066). Follistatin increased satellite cell counts after denervation ( p < 0.001) but reduced satellite cell counts after sham denervation ( p = 0.037). Conclusion Follistatin had mixed effects on muscle weight and force. Direct FST protein delivery enhanced satellite cell counts following reinnervation. The positive effect on the satellite cell population is intriguing and warrants further investigation.

The Role of Satellite Cells in Skeletal Muscle Regeneration-The Effect of Exercise and Age.

Simple SummaryStudies describing the effects of various forms of exercise and age on muscle regeneration were reviewed. Satellite cells are a heterogeneous group of cells that includes stem cells and skeletal muscle progenitor cells. Each skeletal muscle fiber has its own pool of satellite cells that remain inactive until the muscle is damaged. Minor damage within the cell membrane of muscle fibers is patched by fusing intracellular vesicles with the damaged sarcolemma. More severe muscle damage initiates a multistep regeneration process in which satellite cells play an essential role. The condition that initiates the cascade of reactions is the formation of inflammation at the structural discontinuity site, resulting in satellite cell activation. The multitude of reactions and pathways occurring during this process means that many different substances are involved in it and control it. Not all of them are well-understood yet. In parallel, the body’s own population of satellite cells is being rebuilt so that more fibers can be regenerated in the future. Athletes and the elderly are primarily at risk for muscle damage, and pathologies in muscle fiber regeneration cause serious diseases.The population of satellite cells (mSCs) is highly diversified. The cells comprising it differ in their ability to regenerate their own population and differentiate, as well as in the properties they exhibit. The heterogeneity of this group of cells is evidenced by multiple differentiating markers that enable their recognition, classification, labeling, and characterization. One of the main tasks of satellite cells is skeletal muscle regeneration. Myofibers are often damaged during vigorous exercise in people who participate in sports activities. The number of satellite cells and the speed of the regeneration processes that depend on them affect the time structure of an athlete’s training. This process depends on inflammatory cells. The multitude of reactions and pathways that occur during the regeneration process results in the participation and control of many factors that are activated and secreted during muscle fiber damage and at different stages of its regeneration. However, not all of them are well understood yet. This paper presents the current state of knowledge on satellite cell-dependent skeletal muscle regeneration. Studies describing the effects of various forms of exercise and age on this process were reviewed.