By Ng Chi Wai, Jessie
A century ago, it was discovered that skeletal muscles can regenerate new muscle fibres after being damaged. Alexander Mauro was a great contributor to the understanding of this regeneration process; in 1961, he observed mononuclear cells between the basal lamina surrounding each muscle fibre and its plasma membrane and named these cells satellite cells (SCs) (Mauro, 1961). Later on, it was established that SCs are the main players of skeletal muscle regeneration.
In uninjured adult muscles, SCs are quiescent. When muscles are injured, wound-derived signals, including hepatocyte growth factor (HGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF) and nitric oxide (NO) (Tedesco et al., 2010), activate SCs to express myogenic factor 5 (Myf5+) and myoblast determination protein 1 (MyoD+) (Tedesco et al., 2010). Activated SCs are called myoblasts, which proliferate massively to generate myogenic progenitors – the precursors of skeletal muscles (Dumont, Wang and Rudnicki, 2015). Myoblasts then downregulate the expression of Pax7 and upregulate the expression of myogenin (Myog) and Myf6 to leave the cell cycle, differentiate, and fuse together to form myofibers (Dumont, Wang and Rudnicki, 2015).
Injury experiments have shown that SC numbers remain constant, even after various injuries (Shi and Garry, 2006), demonstrating that SCs are able to self-renew. There is also evidence from multiple studies supporting SCs’ heterogeneity and showing that SCs have a subgroup that is more likely to progress into myogenic lineage and another subgroup that is destined to undergo self-renewal.
Other Myogenic Progenitors
Some unorthodox myogenic progenitors outside skeletal muscles have been identified using transgenic markers, such as neural stem cells from ectoderm, hematopoietic, and mesoderm cells (Tedesco et al., 2010). Neural stem cells are the only stem cells derived from ectoderm that can differentiate into skeletal muscles when cultured with skeletal myoblasts or transplanted to the site of skeletal muscle regeneration (Galli et al., 2000).
As for hematopoietic cells, it was established that the CD45+ fraction of the bone marrow has myogenic potential. However, researchers have disagreed on when this differentiation occurs. One study on a mouse model demonstrated that one hematopoietic progenitor can reconstruct the hematopoietic system and can also regenerate at a lower frequency (Corbel et al., 2003), meanwhile, another similar study demonstrated that CD45+ hematopoietic progenitors help to regenerate skeletal muscles by fusing mature myeloid cells instead of hematopoietic stem cells (Camargo et al., 2003). The CD133+ fraction, which is in circulation, expresses early myogenic markers (Torrente et al., 2004). Other fractions of hematopoietic cells which have not been well-characterized seem to have myogenic potency as well, but only at a low frequency.
Aside from hematopoietic cells, other cells from mesoderm have been experimented and shown to have myogenic potential, typically after being treated with drugs, modified genetically or cultured with SCs or myoblasts (Tedesco et al., 2010). However, there are a few exceptions like mesenchymal stem cells, multipotent adult progenitor cells, myeloid-derived suppressor cells, CD133+ cells etc., which have been documented to be able to carry out myogenesis in vivo (Tedesco et al., 2010).
These cells can differentiate to myogenic stem cells like pericytes, myoendothelial cells and interstitial cells, then eventually enter the SC pool and regenerate skeletal muscles (Tedesco et al., 2010).
A groundbreaking discovery of macrophages during skeletal muscle regeneration
Macrophages are specialised myeloid cells derived from monocytes and are involved in the recognition, phagocytosis, destruction of pathogens and fighting off bacterial infections.. Recent studies show that macrophages are also involved in the generation of stem cells in skeletal muscles (Ratnayake et al., 2021).
A muscle injury model in transgenic larval zebrafish was used to study the link between macrophages and skeletal muscle regeneration. The muscles of zebrafish were first injured with laser ablation and the macrophage responses before and after the injury were then documented with multiphoton imaging. It was found that approximately 34% of macrophages that are proximate to the wound migrated towards the lesion (Ratnayake et al., 2021). Meanwhile, the other proximate macrophages did not, which suggested that within the macrophage population, only a specific subset responded to the injury. Among the macrophages that migrated to the injury, around 51.11% remained within the wound 24 hours after the injury had occurred (Ratnayake et al., 2021). Researchers referred to such behaviour as “dwelling”. Interestingly, the macrophages that left the wound exhibited a stellate appearance while the dwelling ones remained spherical – such transition occurred regardless of wound size (Ratnayake et al., 2021).
Establishing whether the dwelling macrophages originated from the response macrophages or a migratory wave induced by other pathways involved using a transgenic line of zebrafish. In these transgenic zebrafishes, the macrophages expressed a photoconvertible fluorescent protein called Kaede. One day after the injury, wound-localized macrophages were photo-converted to distinguish them from macrophages external to the injury. The dwelling macrophages all exhibited photo-converted Kaede one day later, which indicated that they were all derived from the initial transient population (Ratnayake et al., 2021).
Further experiments revealed that the dwelling macrophages established a transient but obligate niche for stem cell proliferation. Single-cell profiling was used to identify proliferative signals that dwelling macrophages secrete. One of the secreted signals was cytokine nicotinamide phosphoribosyltransferase (NAMPT, visfatin or PBEF in humans) (Ratnayake et al., 2021). Besides being important in cellular metabolism and NAD regeneration, NAMPT also alters the expression of myogenic regulatory factors in myoblasts. Moreover, chemokine receptor type 5 (CCR5), which is a putative cell surface receptor for NAMPT expressed on muscle stem cells (MuSCs), was found to be upregulated in a mouse model (Hirata et al., 2003).
To study how NAMPT is involved in myoblast proliferation, mammalian cell culture systems were first used, demonstrating how NAMPT has a high affinity for both mouse and human CCR5 (Ratnayake et al., 2021). Next, mouse cell models were used to see if NAMPT elicits a signalling cascade that encourages proliferation. An inhibitor of NAMPT was used and it had no effect on cell proliferation, which indicates that the pro-proliferative nature of NAMPT is not dependent on its intracellular enzymatic function (Ratnayake et al., 2021). HrNAMPT (human recombinant NAMPT) was able to heighten the proliferation of SCs in myoblasts (Ratnayake et al., 2021). It was also found that SCs proliferated when macrophages were present, but not when co-cultured with other cells. Notably, when macrophages are present, the addition of hrNAMPT did not increase SC proliferation – suggesting that there is a maximum proliferation rate due to receptor saturation (Ratnayake et al., 2021). The supplementation of CCL4, a chemokine specifically binds to CCR5, mirrored the effect of NAMPT supplementation (Ratnayake et al., 2021). It is therefore confirmed that NAMPT induces proliferation via CCR5.
To establish that CCR5 is necessary for MuSCs, Ratnayake et al. mutated ccr5 in MuSCs of transgenic zebrafish larvae and found that the number of dwelling macrophages in the wound and the association between dwelling macrophages and stem cells were not altered (Ratnayake et al., 2021). However, the muscle repair ability and MuSC proliferation of the larvae were significantly impaired after muscle injury (Ratnayake et al., 2021). Then, hrNAMPT was delivered into the muscle defect of a mouse model with volumetric muscle loss and fully restored muscle architecture at the wound site. The delivery of hrNAMPT resulted in an increase in the total number and proportion of proliferated SCs and also the number of centrally nucleated de novo muscle fibres (Ratnayake et al., 2021).
Potential Clinical Applications
The study of skeletal muscle regeneration can help progress new treatments for skeletal muscle injury and disease. It is suggested that emphasis should be put on the specific injury-located macrophages when conducting research on skeletal muscle injury treatments, as the transplantation of isolated Muscle SCs has yet to provide evidence of its therapeutic effects (Ratnayake et al., 2021).
Another possible application is muscle regeneration by embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) via mesodermal progenitors (Tedesco et al., 2010). A benefit of this approach is being able to deliver myogenic progenitors through blood circulation. It has been demonstrated that deriving mesodermal progenitors from mouse ESCs has contributed to muscle regeneration after transplantation (Tedesco et al., 2010). However, there are safety concerns, such as tumour formation, that need to be tackled before putting the concept into practice (Tedesco et al., 2010). Standardised protocols for the generation of iPSCs and stringent tumorigenic assays for the derived cell types must be used before proceeding to clinical application (Tedesco et al., 2010).
As the role of myogenic progenitors in skeletal muscle regeneration becomes clearer, there is increasing interest in the potential of regenerative clinical applications. This progression will require greater consideration of patient safety and close consultation with patients to evaluate any possible risks. Despite these challenges, research in the field of myogenic progenitors has captured global interest, and has brought great excitement to the field of regenerative medicine.
Saldana, J.I. (2016). Macrophages | British Society for Immunology. [online] Immunology.org. Available at: https://www.immunology.org/public-information/bitesized-immunology/cells/macrophages.
Bah, A. and Vergne, I. (2017). Macrophage Autophagy and Bacterial Infections. Frontiers in Immunology, [online] 8. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5681717/ [Accessed 1 Mar. 2020].
Ratnayake, D., Nguyen, P.D., Rossello, F.J., Wimmer, V.C., Tan, J.L., Galvis, L.A., Julier, Z., Wood, A.J., Boudier, T., Isiaku, A.I., Berger, S., Oorschot, V., Sonntag, C., Rogers, K.L., Marcelle, C., Lieschke, G.J., Martino, M.M., Bakkers, J. and Currie, P.D. (2021). Macrophages provide a transient muscle stem cell niche via NAMPT secretion. Nature, [online] pp.1–7. Available at: https://www.nature.com/articles/s41586-021-03199-7.pdf [Accessed 21 Feb. 2021].
Mauro, A. (1961). SATELLITE CELL OF SKELETAL MUSCLE FIBERS. The Journal of Cell Biology, 9(2), pp.493–495.
Tedesco, F.S., Dellavalle, A., Diaz-Manera, J., Messina, G. and Cossu, G. (2010). Repairing skeletal muscle: regenerative potential of skeletal muscle stem cells. Journal of Clinical Investigation, 120(1), pp.11–19.
Shi, X. and Garry, D.J. (2006). Muscle stem cells in development, regeneration, and disease. Genes & Development, 20(13), pp.1692–1708.
Hirata, A., Masuda, S., Tamura, T., Kai, K., Ojima, K., Fukase, A., Motoyoshi, K., Kamakura, K., Miyagoe-Suzuki, Y. and Takeda, S. (2003). Expression Profiling of Cytokines and Related Genes in Regenerating Skeletal Muscle after Cardiotoxin Injection. The American Journal of Pathology, 163(1), pp.203–215.
Watanabe, R., Hilhorst, M., Zhang, H., Zeisbrich, M., Berry, G.J., Wallis, B.B., Harrison, D.G., Giacomini, J.C., Goronzy, J.J. and Weyand, C.M. (2018). Glucose metabolism controls disease-specific signatures of macrophage effector functions. JCI insight, [online] 3(20). Available at: https://pubmed.ncbi.nlm.nih.gov/30333306/ [Accessed 21 Feb. 2021].
Galli, R., Borello, U., Gritti, A., Minasi, M.G., Bjornson, C., Coletta, M., Mora, M., De Angelis, M.G., Fiocco, R., Cossu, G. and Vescovi, A.L. (2000). Skeletal myogenic potential of human and mouse neural stem cells. Nature Neuroscience, [online] 3(10), pp.986–991. Available at: https://pubmed.ncbi.nlm.nih.gov/11017170/ [Accessed 24 Feb. 2021].
Corbel, S.Y., Lee, A., Yi, L., Duenas, J., Brazelton, T.R., Blau, H.M. and Rossi, F.M.V. (2003). Contribution of hematopoietic stem cells to skeletal muscle. Nature Medicine, [online] 9(12), pp.1528–1532. Available at: https://pubmed.ncbi.nlm.nih.gov/14625543/ [Accessed 25 Feb. 2021].
Torrente, Y., Belicchi, M., Sampaolesi, M., Pisati, F., Meregalli, M., D’Antona, G., Tonlorenzi, R., Porretti, L., Gavina, M., Mamchaoui, K., Pellegrino, M.A., Furling, D., Mouly, V., Butler-Browne, G.S., Bottinelli, R., Cossu, G. and Bresolin, N. (2004). Human circulating AC133(+) stem cells restore dystrophin expression and ameliorate function in dystrophic skeletal muscle. The Journal of Clinical Investigation, [online] 114(2), pp.182–195. Available at: https://pubmed.ncbi.nlm.nih.gov/15254585/ [Accessed 25 Feb. 2021].
Camargo, F.D., Green, R., Capetanaki, Y., Jackson, K.A., Goodell, M.A. and Capetenaki, Y. (2003). Single hematopoietic stem cells generate skeletal muscle through myeloid intermediates. Nature Medicine, [online] 9(12), pp.1520–1527. Available at: https://pubmed.ncbi.nlm.nih.gov/14625546/ [Accessed 25 Feb. 2021].