Most organisms experience changes in regenerative abilities through their lifespan. During aging, numerous tissues exhibit a progressive decline in homeostasis and regeneration that results in tissue degeneration, malfunction and pathology. The mechanisms responsible for this decay are both cell intrinsic, such as cellular senescence, as well as cell-extrinsic, such as changes in the regenerative environment. Understanding how these mechanisms impact on regenerative processes is essential to devise therapeutic approaches to improve tissue regeneration and extend healthspan. This review offers an overview of how regenerative abilities change through lifespan in various organisms, the factors that underlie such changes and the avenues for therapeutic intervention. It focuses on established models of mammalian regeneration as well as on models in which regenerative abilities do not decline with age, as these can deliver valuable insights for our understanding of the interplay between regeneration and aging.

Changes in Regenerative Capacity through Lifespan.

Due to the considerable environmental impact and the controversial animal welfare associated with industrial meat production, combined with the ever-increasing global population and demand for meat products, sustainable production alternatives are indispensable. In 2013, the world's first laboratory grown hamburger made from cultured muscle cells was developed. However, coming at a price of $300.000, and being produced manually, substantial effort is still required to reach sustainable large-scale production. One of the main challenges is scalability. Microcarriers (MCs), offering a large surface/volume ratio, are the most promising candidates for upscaling muscle cell culture. However, although many MCs have been developed for cell lines and stem cells typically used in the medical field, none have been specifically developed for muscle stem cells and meat production. This paper aims to discuss the MCs' design criteria for skeletal muscle cell proliferation and subsequently for meat production based on three scenarios: (1) MCs are serving only as a temporary substrate for cell attachment and proliferation and therefore they need to be separated from the cells at some stage of the bioprocess, (2) MCs serve as a temporary substrate for cell proliferation but are degraded or dissolved during the bioprocess, and (3) MCs are embedded in the final product and therefore need to be edible. The particularities of each of these three bioprocesses will be discussed from the perspective of MCs as well as the feasibility of a one-step bioprocess. Each scenario presents advantages and drawbacks, which are discussed in detail, nevertheless the third scenario appears to be the most promising one for a production process. Indeed, using an edible material can limit or completely eliminate dissociation/degradation/separation steps and even promote organoleptic qualities when embedded in the final product. Edible microcarriers could also be used as a temporary substrate similarly to scenarios 1 and 2, which would limit the risk of non-edible residues.

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Microcarriers for Upscaling Cultured Meat Production.

Skeletal muscle contains a designated population of adult stem cells, called satellite cells, which are generally quiescent. In homeostasis, satellite cells proliferate only sporadically and usually by asymmetric cell division to replace myofibres damaged by daily activity and maintain the stem cell pool. However, satellite cells can also be robustly activated upon tissue injury, after which they undergo symmetric divisions to generate new stem cells and numerous proliferating myoblasts that later differentiate to muscle cells (myocytes) to rebuild the muscle fibre, thereby supporting skeletal muscle regeneration. Recent discoveries show that satellite cells have a great degree of population heterogeneity, and that their cell fate choices during the regeneration process are dictated by both intrinsic and extrinsic mechanisms. Extrinsic cues come largely from communication with the numerous distinct stromal cell types in their niche, creating a dynamically interactive microenvironment. This Review discusses the role and regulation of satellite cells in skeletal muscle homeostasis and regeneration. In particular, we highlight the cell-intrinsic control of quiescence versus activation, the importance of satellite cell–niche communication, and deregulation of these mechanisms associated with ageing. The increasing understanding of how satellite cells are regulated will help to advance muscle regeneration and rejuvenation therapies. Satellite cells are skeletal muscle stem cells that are largely quiescent. They are activated upon muscle damage and differentiate into muscle cells or return to quiescence. These processes are controlled by cell-intrinsic mechanisms and by signals from the niche, and are deregulated in ageing, leading to impaired muscle regeneration.

Control of satellite cell function in muscle regeneration and its disruption in ageing.

Mechanosensing of matrix by stem cells: From matrix heterogeneity, contractility, and the nucleus in pore-migration to cardiogenesis and muscle stem cells in vivo

Muscle cells sense the mechanical properties of their microenvironment, and these properties can change in response to injury or disease. Hydrogels with dynamic material properties can be used to study the effect of such varying mechanical signals. Here, we report the ability of azadibenzocyclooctyne to undergo a cytocompatible, photoinitiated crosslinking reaction. This reaction is exploited as a strategy for on-demand stiffening of three-dimensional cell scaffolds formed through an initial strain-promoted azide-alkyne cycloaddition. Myoblasts encapsulated in these networks respond to increased matrix stiffness through decreased cell spreading and nuclear localization of Yes-associated protein 1 (YAP). However, when the photocrosslinking reaction is delayed to allow cell spreading, elongated myoblasts display increased YAP nuclear localization.

Secondary Photocrosslinking of Click Hydrogels To Probe Myoblast Mechanotransduction in Three Dimensions

Background 
Skeletal muscle aging is associated with a decreased regenerative potential due to the loss of function of endogenous stem cells or myogenic progenitor cells (MPCs). Aged skeletal muscle is characterized by the deposition of extracellular matrix (ECM), which in turn influences the biomechanical properties of myofibers by increasing their stiffness. Since the stiffness of the MPC microenvironment directly impacts MPC function, we hypothesized that the increase in muscle stiffness that occurs with aging impairs the behavior of MPCs, ultimately leading to a decrease in regenerative potential.


Results 
We showed that freshly isolated individual myofibers from aged mouse muscles contain fewer MPCs overall than myofibers from adult muscles, with fewer quiescent MPCs and more proliferative and differentiating MPCs. We observed alterations in cultured MPC behavior in aged animals, where the proliferation and differentiation of MPCs were lower and higher, respectively. These alterations were not linked to the intrinsic properties of aged myofibers, as shown by the similar values for the cumulative population-doubling values and fusion indexes. However, atomic force microscopy (AFM) indentation experiments revealed a nearly 4-fold increase in the stiffness of the MPC microenvironment. We further showed that the increase in stiffness is associated with alterations to muscle ECM, including the accumulation of collagen, which was correlated with higher hydroxyproline and advanced glycation end-product content. Lastly, we recapitulated the impaired MPC behavior observed in aging using a hydrogel substrate that mimics the stiffness of myofibers.


Conclusions 
These findings provide novel evidence that the low regenerative potential of aged skeletal muscle is independent of intrinsic MPC properties but is related to the increase in the stiffness of the MPC microenvironment.

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Increased Stiffness in Aged Skeletal Muscle Impairs Muscle Progenitor Cell Proliferative Activity

[This corrects the article DOI: 10.1371/journal.pone.0136217.].

/pdf/correction-increased-stiffness-in-aged-skeletal-muscle-3fe19m2398.pdf

Noémie Veillette

Papers

Increased Stiffness in Aged Skeletal Muscle Impairs Muscle Progenitor Cell Proliferative Activity

Correction: Increased Stiffness in Aged Skeletal Muscle Impairs Muscle Progenitor Cell Proliferative Activity

Speeding up the Production of Clinical-Grade Skin Substitutes Using Off-the-shelf Decellularized Self-Assembled Dermal Matrices.