Comparative analysis of cattle breeds as satellite cells donors for cultured beef

Background: Cultured meat is a promising new field with the potential for considerable environmental and animal welfare benefits. One technological approach to cultured meat production utilises the proliferative and differentiative capacity of muscle-derived satellite cells (SCs) to produce large volumes of cultured muscle tissue from small biopsies of donor animals. Differing genotypes between cattle breeds lead to predictable phenotypic traits, resulting in breeds being favoured for their respective meat or milk production characteristics in the livestock industry. However, whilst these breeds show significant differences in muscle growth, it is unclear whether the physiological differences observed between them in vivo are reflected in differences in SC behaviour in vitro, particularly with respect to proliferation, differentiation and cellular longevity, and hence whether particular breeds might represent preferred SC donors for a cultured beef bioprocess. Results: Comparing SCs isolated from five breeds (Belgian Blue, Holstein Friesian, Galloway, Limousin and Simmental), we found that the proliferation rates were largely unaffected by the donor breed. In contrast, potentially meaningful differences were observed in the kinetics and extent of myogenic differentiation. Furthermore, whilst differentiation dropped for all breeds with increasing population doublings (PDs), SCs from Belgian Blue and Limousin cattle showed significantly longer retention of differentiation capacity over long-term passaging. Conclusion: SCs from all breeds were able to proliferate and differentiate, although Limousin and (particularly) Belgian Blue cattle, both breeds commonly used for traditional meat production, may represent preferred donors for cultured beef production.

Thermal stress affects proliferation and differentiation of turkey satellite cells through the mTOR/S6K pathway in a growth-dependent manner

Satellite cells (SCs) are stem cells responsible for post-hatch muscle growth through hypertrophy and in birds are sensitive to thermal stress during the first week after hatch. The mechanistic target of rapamycin (mTOR) signaling pathway, which is highly responsive to thermal stress in differentiating turkey pectoralis major (p. major) muscle SCs, regulates protein synthesis and the activities of SCs through a downstream effector, S6 kinase (S6K). The objectives of this study were: 1) to determine the effect of heat (43°C) and cold (33°C) stress on activity of the mTOR/S6K pathway in SCs isolated from the p. major muscle of one-week-old faster-growing modern commercial (NC) turkeys compared to those from slower-growing Randombred Control Line 2 (RBC2) turkeys, and 2) to assess the effect of mTOR knockdown on the proliferation, differentiation, and expression of myogenic regulatory factors of the SCs. Heat stress increased phosphorylation of both mTOR and S6K in both turkey lines, with greater increases observed in the RBC2 line. With cold stress, greater reductions in mTOR and S6K phosphorylation were observed in the NC line. Early knockdown of mTOR decreased proliferation, differentiation, and expression of myoblast determination protein 1 and myogenin in both lines independent of temperature, with the RBC2 line showing greater reductions in proliferation and differentiation than the NC line at 38° and 43°C. Proliferating SCs are more dependent on mTOR/S6K-mediated regulation than differentiating SCs. Thus, thermal stress can affect breast muscle hypertrophic potential by changing satellite cell proliferation and differentiation, in part, through the mTOR/S6K pathway in a growth-dependent manner. These changes may result in irreversible effects on the development and growth of the turkey p. major muscle.

Skeletal muscle regeneration is altered in the R6/2 mouse model of Huntington's disease

Huntington's disease (HD) is caused by CAG repeat expansion in the huntingtin (HTT) gene. Skeletal muscle wasting alongside central pathology is a well-recognized phenomenon seen in patients with HD and HD mouse models. HD muscle atrophy progresses with disease and affects prognosis and quality of life. Satellite cells, progenitors of mature skeletal muscle fibers, are essential for proliferation, differentiation, and repair of muscle tissue in response to muscle injury or exercise. In this study, we aim to investigate the effect of mutant HTT on the differentiation and regeneration capacity of HD muscle by employing in vitro mononuclear skeletal muscle cell isolation and in vivo acute muscle damage model in R6/2 mice. We found that, similar to R6/2 adult mice, neonatal R6/2 mice also exhibit a significant reduction in myofiber width and morphological changes in gastrocnemius and soleus muscles compared to WT mice. Cardiotoxin (CTX)-induced acute muscle damage in R6/2 and WT mice showed that the Pax7+ satellite cell pool was dampened in R6/2 mice at 4 weeks post-injection, and R6/2 mice exhibited an altered inflammatory profile in response to acute damage. Our results suggest that, in addition to the mutant HTT degenerative effects in mature muscle fibers, expression of mutant HTT in satellite cells might alter developmental and regenerative processes to contribute to the progressive muscle mass loss in HD. Taken together, the results presented here encourage further studies evaluating the underlying mechanisms of satellite cell dysfunction in HD mouse models.

FHL2 Regulates Proliferation Differentiation and Autophagy of Bovine Skeletal Muscle Satellite Cells Through Wnt/β-catenin Signaling Pathway

The mechanism of physiological regulation of bovine skeletal muscle development is a complex process, and FHL2 has been studied in association with β-catenin activity and has previously been reported to play a role in skeletal muscle.However the mechanism of FHL2 action in regulating skeletal muscle development in bovine skeletal myosatellite is unclear. Here, we report that FHL2 can both promote the proliferation and differentiation of bovine myosatellite cells through the wnt signaling pathway and bovine skeletal muscle satellite cells through cellular autophagy. The results of western blotting, rt-qPCT, cell transfection assay showed that FHL2 gene expression was enhanced during the proliferation of skeletal muscle satellite cells, and FHL2 knockdown inhibited the proliferation and differentiation of bovine satellite cells and promoted the atrophy of myotubes. Furthermore, immunoprecipitation assays yielded that FHL2 knockdown decreased β-catenin activity in BMSCs and activated β-catenin-mediated wnt signaling pathway in combination with DVL-2, and that FHL2 knockdown induced autophagy in bovine satellite cells. Therefore, the FHL2 gene is a key gene in the regulation of bovine satellite cells.

Satellite Cells Exhibit Decreased Numbers and Impaired Functions on Single Myofibers Isolated from Vitamin B6-Deficient Mice

Emerging research in human studies suggests an association among vitamin B6, sarcopenia, and muscle strength. However, very little is known regarding its potential role at the cellular level, especially in muscle satellite cells. Therefore, to determine whether vitamin B6 affects the satellite cells, we isolated single myofibers from muscles of vitamin B6-deficient and vitamin B6-supplemented mice. Subsequently, we subjected them to single myofiber culture and observed the number and function of the satellite cells, which remained in their niche on the myofibers. Prior to culture, the vitamin B6-deficient myofibers exhibited a significantly lower number of quiescent satellite cells, as compared to that in the vitamin B6-supplemented myofibers, thereby suggesting that vitamin B6 deficiency induces a decline in the quiescent satellite cell pool in mouse muscles. After 48 and 72 h of culture, the number of proliferating satellite cells per cluster was similar between the vitamin B6-deficient and -supplemented myofibers, but their numbers decreased significantly after culturing the myofibers in vitamin B6-free medium. After 72 h of culture, the number of self-renewing satellite cells per cluster was significantly lower in the vitamin B6-deficient myofibers, and the vitamin B6-free medium further decreased this number. In conclusion, vitamin B6 deficiency appears to reduce the number of quiescent satellite cells and suppress the proliferation and self-renewal of satellite cells during myogenesis.

Tributyrin, a Butyrate Pro-Drug, Primes Satellite Cells for Differentiation by Altering the Epigenetic Landscape

Satellite cells (SC) are a population of muscle resident stem cells that are responsible for postnatal muscle growth and repair. With investigation into the genomic regulation of SC fate, the role of the epigenome in governing SC myogenesis is becoming clearer. Histone deacetylase (HDAC) inhibitors have been demonstrated to be effective at enhancing the myogenic program of SC, but their role in altering the epigenetic landscape of SC remains undetermined. Our objective was to determine how an HDAC inhibitor, butyrate, promotes myogenic differentiation. SC from tributyrin treated neonatal piglets showed a decrease relative to SC from control animals in the expression of enhance of zeste homologue-2 (EZH2), a chromatin modifier, ex vivo. Chromatin Immunoprecipitation-Sequencing (ChIP-Seq) analysis of SC isolated from tributyrin treated pigs showed a global reduction of the tri-methylation of lysine 27 of histone H3 (H3K27me3) repressive chromatin mark. To determine if reductions in EZH2 was the primary mechanism through which butyrate affects SC behavior, SC were transfected with siRNA targeting EZH2, treated with 0.5 mM butyrate, or both. Treatment with butyrate reduced paired-box-7 (Pax7) and myogenic differentiation-1 (MyoD) gene expression, while siRNA caused reductions in EZH2 had no effect on their expression. EZH2 depletion did result in an increase in differentiating SC, but not in myotube hypertrophy. These results indicate that while EZH2 reduction may force myogenic differentiation, butyrate may operate through a parallel mechanism to enhance the myogenic program.

Available In Vitro Models for Human Satellite Cells from Skeletal Muscle

Skeletal muscle accounts for almost 40% of the total adult human body mass. This tissue is essential for structural and mechanical functions such as posture, locomotion, and breathing, and it is endowed with an extraordinary ability to adapt to physiological changes associated with growth and physical exercise, as well as tissue damage. Moreover, skeletal muscle is the most age-sensitive tissue in mammals. Due to aging, but also to several diseases, muscle wasting occurs with a loss of muscle mass and functionality, resulting from disuse atrophy and defective muscle regeneration, associated with dysfunction of satellite cells, which are the cells responsible for maintaining and repairing adult muscle. The most established cell lines commonly used to study muscle homeostasis come from rodents, but there is a need to study skeletal muscle using human models, which, due to ethical implications, consist primarily of in vitro culture, which is the only alternative way to vertebrate model organisms. This review will survey in vitro 2D/3D models of human satellite cells to assess skeletal muscle biology for pre-clinical investigations and future directions.

Genome-wide identification of enhancers and transcription factors regulating the myogenic differentiation of bovine satellite cells

Background Satellite cells are the myogenic precursor cells in adult skeletal muscle. The objective of this study was to identify enhancers and transcription factors that regulate gene expression during the differentiation of bovine satellite cells into myotubes. Results Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) was performed to identify genomic regions where lysine 27 of H3 histone is acetylated (H3K27ac), i.e., active enhancers, from bovine satellite cells before and during differentiation into myotubes. A total of 19,027 and 47,669 H3K27ac-marked enhancers were consistently identified from two biological replicates of before- and during-differentiation bovine satellite cells, respectively. Of these enhancers, 5882 were specific to before-differentiation, 35,723 to during-differentiation, and 13,199 common to before- and during-differentiation bovine satellite cells. Whereas most of the before- or during-differentiation-specific H3K27ac-marked enhancers were located distally to the transcription start site, the enhancers common to before- and during-differentiation were located both distally and proximally to the transcription start site. The three sets of H3K27ac-marked enhancers were associated with functionally different genes and enriched with different transcription factor binding sites. Specifically, many of the H3K27ac-marked enhancers specific to during-differentiation bovine satellite cells were associated with genes involved in muscle structure and development, and were enriched with binding sites for the MyoD, AP-1, KLF, TEAD, and MEF2 families of transcription factors. A positive role was validated for Fos and FosB, two AP-1 family transcription factors, in the differentiation of bovine satellite cells into myotubes by siRNA-mediated knockdown. Conclusions Tens of thousands of H3K27ac-marked active enhancers have been identified from bovine satellite cells before or during differentiation. These enhancers contain binding sites not only for transcription factors whose role in satellite cell differentiation is well known but also for transcription factors whose role in satellite cell differentiation is unknown. These enhancers and transcription factors are valuable resources for understanding the complex mechanism that mediates gene expression during satellite cell differentiation. Because satellite cell differentiation is a key step in skeletal muscle growth, the enhancers, the transcription factors, and their target genes identified in this study are also valuable resources for identifying and interpreting skeletal muscle trait-associated DNA variants in cattle.

Epigenetic Evidence for Distinct Contributions of Resident and Acquired Myonuclei During Long-Term Exercise Adaptation Using Timed In Vivo Myonuclear Labeling

Muscle fibers are syncytial post-mitotic cells that can acquire exogenous nuclei from resident muscle stem cells, called satellite cells. Myonuclei are added to muscle fibers by satellite cells during conditions such as load-induced hypertrophy. It is difficult to dissect the molecular contributions of resident versus satellite cell-derived myonuclei during adaptation due to the complexity of labeling distinct nuclear populations in multinuclear cells without label transference between nuclei. To sidestep this barrier, we utilized a genetic mouse model where myonuclear DNA can be specifically and stably labeled via non-constitutive H2B-GFP at any point in the lifespan. Resident myonuclei (Mn) were GFP-tagged in vivo before eight weeks of progressive weighted wheel running (PoWeR) in adult mice (>4-month-old). Resident+satellite cell-derived myonuclei (Mn+SC Mn) were labeled at the end of PoWeR in a separate cohort. Following myonuclear isolation, promoter DNA methylation profiles acquired with low-input RRBS were compared to deduce epigenetic contributions of satellite cell-derived myonuclei during adaptation. Resident myonuclear DNA has hypomethylated promoters in genes related to protein turnover, while the addition of satellite cell-derived myonuclei shifts myonuclear methylation profiles to favor transcription factor regulation and cell-cell signaling. By comparing myonucleus-specific methylation profiling to previously published single-nucleus transcriptional analysis in the absence (Mn) versus presence of satellite cells (Mn+SC Mn) with PoWeR, we provide evidence that satellite cell-derived myonuclei may preferentially supply ribosomal proteins to growing myofibers and retain an epigenetic "memory" of prior stem cell identity. These data offer insights on distinct epigenetic myonuclear characteristics and contributions during adult muscle growth.