Evaluating clock properties in skeletal muscle, this chapter uses the Per2Luc reporter line as the gold standard method. This method proves useful in assessing clock function in ex vivo muscle preparations, employing a range of samples including intact muscle groups, dissected muscle strips, and primary myoblast or myotube cell cultures.
Regenerative models of muscle have exposed the intricacies of inflammatory responses, the removal of damaged tissue, and the targeted repair orchestrated by stem cells, ultimately benefiting therapeutic approaches. Although the most advanced muscle repair research is performed using rodents, zebrafish are now presenting themselves as a significant alternative model system, leveraging both genetic and optical characteristics. Several publications have discussed protocols for inducing muscle injury, employing both chemical and physical mechanisms. Zebrafish larval skeletal muscle regeneration across two stages is investigated using simple, inexpensive, precise, adaptable, and efficient wounding and analytical techniques. We present case studies of the individual larval response to muscle damage, the subsequent ingress of muscle stem cells, the involvement of immune cells, and the subsequent fiber regeneration, all tracked over an extended timeframe. These analyses could substantially improve our comprehension by reducing the reliance on averaging regeneration responses across individuals who are inevitably exposed to varying wound stimuli.
Denervating the skeletal muscle in rodents produces the nerve transection model, a well-established and validated experimental model of skeletal muscle atrophy. Whilst many denervation methods exist in rats, the development of multiple transgenic and knockout mouse lines has greatly increased the application of mouse models in nerve transection studies. Studies involving skeletal muscle denervation are instrumental in expanding our comprehension of how nerve activity and/or neurotrophic substances influence the ability of skeletal muscles to change. In mice and rats, the sciatic or tibial nerve is frequently denervated experimentally, as resection of these nerves is relatively straightforward. Recent publications frequently detail experiments involving tibial nerve transection in mice. The procedures for severing the sciatic and tibial nerves in mice are demonstrated and explained in this chapter.
The highly plastic nature of skeletal muscle allows it to modify its mass and strength in response to mechanical stimulation, including overloading and unloading, which correspondingly lead to the processes of hypertrophy and atrophy. The interplay of mechanical loading within the muscle and muscle stem cell dynamics, including activation, proliferation, and differentiation, is complex. Bio-imaging application While experimental models of mechanical loading and unloading have been extensively employed to examine the molecular underpinnings of muscular plasticity and stem cell function, detailed descriptions of these methods remain scarce in the literature. This paper details the necessary steps for inducing tenotomy-induced mechanical overload and tail-suspension-induced mechanical unloading, two of the most common and simplest techniques for inducing muscle hypertrophy and atrophy in mouse studies.
To adapt to fluctuating physiological and pathological settings, skeletal muscle employs either myogenic progenitor cell regeneration or modifications to muscle fiber characteristics, metabolic processes, and contractile capacities. buy FLT3-IN-3 In order to analyze these transformations, suitable muscle tissue samples must be prepared. Subsequently, the need for reliable methods to analyze and evaluate skeletal muscle characteristics is apparent. However, even with enhancements in the technical procedures for genetic investigation of skeletal muscle, the core strategies for identifying muscle pathologies have remained static over many years. Hematoxylin and eosin (H&E) staining and antibody procedures are fundamental and commonly employed methodologies to assess skeletal muscle phenotypes. This chapter explores fundamental techniques and protocols for inducing skeletal muscle regeneration, including chemical and cellular transplantation approaches, as well as methods for preparing and evaluating skeletal muscle samples.
The prospect of generating engraftable skeletal muscle progenitor cells provides a compelling cell therapy strategy for combating muscle degeneration. The exceptional proliferative capacity and versatility in differentiation into a multitude of cell lineages make pluripotent stem cells (PSCs) an ideal source for cellular therapies. While ectopic overexpression of myogenic transcription factors and growth factor-driven monolayer differentiation can effectively induce skeletal myogenic lineage development from pluripotent stem cells in a controlled laboratory environment, the resulting muscle cells often lack the reliable engraftment properties required for successful transplantation. A novel method for converting mouse pluripotent stem cells to skeletal myogenic progenitors is presented, circumventing both genetic modification and the necessity for monolayer culture. Through the construction of a teratoma, we routinely collect skeletal myogenic progenitors. Within the limb muscle of an immunocompromised mouse, we initially implant mouse pluripotent stem cells. Using fluorescent-activated cell sorting, 7-integrin and VCAM-1 positive skeletal myogenic progenitors are isolated and purified within a period of three to four weeks. The engraftment efficiency of these teratoma-derived skeletal myogenic progenitors is examined by transplanting them into dystrophin-deficient mice. A teratoma-driven formation process effectively produces skeletal myogenic progenitors with potent regenerative properties from pluripotent stem cells (PSCs), free from genetic alterations or exogenous growth factors.
A sphere-based culture method forms the basis of this protocol, detailing the derivation, maintenance, and differentiation of human pluripotent stem cells into skeletal muscle progenitor/stem cells (myogenic progenitors). Progenitor cell preservation is effectively achieved through sphere-based cultures, owing to their extended lifespans and the vital roles of intercellular communications and signaling molecules. Immune receptor Cellular expansion using this method is a considerable undertaking that proves instrumental for the development of cell-based tissue models and contributes to regenerative medicine's progress.
The majority of muscular dystrophies are directly attributable to genetic conditions. Currently, the only available treatment for these progressive conditions is palliative therapy, as there are no other effective treatments. For the treatment of muscular dystrophy, muscle stem cells are recognized for their potent regenerative and self-renewal capabilities. Anticipated as a potential source for muscle stem cells, human-induced pluripotent stem cells possess an inherent capacity for infinite proliferation and reduced immune reactivity. Even though hiPSC-derived engraftable MuSCs are achievable, their production remains a challenging process due to low efficiency and lack of reproducibility. This protocol, which avoids transgenes, describes how hiPSCs develop into fetal MuSCs, marked by their MYF5 expression. Flow cytometry results, obtained after 12 weeks of differentiation, indicated the presence of roughly 10% of MYF5-positive cells. Analysis of MYF5-positive cells via Pax7 immunostaining indicated that approximately 50-60 percent showed a positive identification. The anticipated utility of this differentiation protocol extends beyond the development of cell therapy, encompassing future breakthroughs in drug discovery utilizing patient-derived induced pluripotent stem cells.
The uses of pluripotent stem cells are manifold, including modeling diseases, evaluating drug efficacy, and providing cell-based therapies for genetic diseases, such as the various forms of muscular dystrophies. Through the application of induced pluripotent stem cell technology, disease-specific pluripotent stem cells can be easily derived for any patient. For the successful deployment of these applications, the targeted in vitro specialization of pluripotent stem cells into muscle cells is critical. Employing transgenes to conditionally express PAX7, a myogenic progenitor population is effectively derived. This population is both expandable and homogeneous, and thus suitable for diverse applications, including in vitro and in vivo studies. Conditional PAX7 expression forms the basis of this optimized protocol for the derivation and expansion of myogenic progenitors from pluripotent stem cells. Our work also includes a detailed description of a more efficient procedure for the terminal differentiation of myogenic progenitors into more mature myotubes, which are better suited for in vitro disease modeling and drug screening applications.
Skeletal muscle interstitial space harbors mesenchymal progenitors, which are critical contributors to pathologies such as fat infiltration, fibrosis, and heterotopic ossification. The contributions of mesenchymal progenitors reach beyond their pathological functions to encompass essential roles in muscle regeneration and the maintenance of muscle homeostasis. Accordingly, thorough and exact analyses of these progenitors are critical for research concerning muscle diseases and optimal health. This method outlines the purification of mesenchymal progenitors using fluorescence-activated cell sorting (FACS), specifically targeting cells expressing the well-established and characteristic PDGFR marker. Cell culture, cell transplantation, and gene expression analysis are just a few of the downstream experiments that can be performed using purified cells. We also describe, using tissue clearing, the process for whole-mount, three-dimensional imaging of mesenchymal progenitors. The methods described within enable a strong foundation for investigating mesenchymal progenitors in skeletal muscle.
Adult skeletal muscle, a dynamic tissue capable of quite efficient regeneration, owes its ability to the presence of its stem cell apparatus. Adult myogenesis is influenced not only by activated satellite cells in response to damage or paracrine factors, but also by other stem cells, acting either directly or indirectly.