The Per2Luc reporter line, the gold standard, is described in this chapter for its application in assessing clock properties of skeletal muscle. This technique is appropriate for the investigation of clock function within ex vivo muscle preparations, utilizing intact muscle groups, dissected muscle strips, and cell culture systems, incorporating primary myoblasts or myotubes.

Studies on muscle regeneration have unveiled the inflammatory cascade, the process of wound clearance, and the stem cell-driven repair of tissue damage, thereby contributing to improved therapies. While rodent studies on muscle repair are highly developed, zebrafish are gaining prominence as an alternative model organism, leveraging both genetic and optical strengths. The literature contains a diversity of muscle-wounding protocols, ranging from chemical to physical interventions. 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. The potential of these analyses is to markedly increase comprehension, by diminishing the requirement to average regeneration responses in individuals encountering a significantly variable wound stimulus.

Denervating the skeletal muscle in rodents produces the nerve transection model, a well-established and validated experimental model of skeletal muscle atrophy. Numerous denervation procedures are employed in rat research, however, the generation of transgenic and knockout mice has also prompted a significant increase in the use of mouse models in nerve transection studies. Skeletal muscle denervation experiments contribute significantly to our knowledge of the crucial influence of nerve signaling and/or neurotrophic components on the plasticity of muscle tissue. Experimental denervation of the sciatic or tibial nerve is a widely used procedure in both mice and rats, as these nerves can be readily resected. Reports on experiments utilizing a tibial nerve transection procedure in mice are appearing with increasing frequency. We demonstrate and elaborate upon the steps taken to transect the sciatic and tibial nerves in mice in this chapter.

Mechanical stimulation, encompassing overloading and unloading, prompts the highly adaptable skeletal muscle tissue to adjust its mass and strength, resulting in hypertrophy or atrophy, respectively. Muscle stem cells' response, including activation, proliferation, and differentiation, is contingent upon the mechanical stress conditions present in the muscle. Primary immune deficiency Experimental models employing mechanical loading and unloading, frequently used to explore the molecular mechanisms of muscle plasticity and stem cell function, are often under-reported with respect to detailed methodologies. 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.

Using myogenic progenitor cells or modifying muscle fiber size, type, metabolic function, and contractile capability, skeletal muscle can respond to shifts in physiological or pathological surroundings. SKL2001 solubility dmso To scrutinize these developments, the preparation of muscle samples must be executed with precision. Consequently, the need for validated methodologies for assessing and evaluating skeletal muscle attributes is crucial. Nonetheless, while the technical tools for genetic analysis of skeletal muscle are enhancing, the primary strategies for detecting muscle abnormalities have persisted over the course of many decades. Skeletal muscle phenotypes are assessed using the straightforward and standardized methodologies of hematoxylin and eosin (H&E) staining or utilizing specific antibodies. 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 creation of engraftable skeletal muscle progenitor cells holds considerable promise for treating muscle diseases marked by degeneration. Pluripotent stem cells' (PSCs) unparalleled ability to proliferate endlessly and differentiate into a wide array of cell types positions them as an ideal cellular source for therapeutic interventions. Despite their in vitro success in converting pluripotent stem cells into skeletal muscle tissue through ectopic overexpression of myogenic transcription factors and growth factor-directed monolayer differentiation, these methods often fall short in producing muscle cells suitable for reliable engraftment after transplantation. We introduce a groundbreaking approach for differentiating mouse pluripotent stem cells into skeletal myogenic progenitors, eschewing genetic alterations and monolayer cultivation. The process of forming a teratoma provides a consistent source of skeletal myogenic progenitors. The procedure begins with the injection of mouse pluripotent stem cells directly into the muscle tissue of an immunocompromised mouse's limb. 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. To assess the effectiveness of engraftment, we subsequently transplant these teratoma-derived skeletal myogenic progenitors 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 approach is used in this protocol for the derivation, maintenance, and differentiation of human pluripotent stem cells into skeletal muscle progenitor/stem cells (myogenic progenitors). Due to their extended lifespan and the significance of cell-cell interactions and signaling molecules, a sphere-based culture method is a suitable approach for progenitor cell maintenance. atypical mycobacterial infection Culture expansion of large numbers of cells is achievable using this method, a significant contribution to the advancement of cellular tissue modeling and regenerative medicine applications.

The genesis of most muscular dystrophies is often linked to genetic irregularities. Effective treatments for these progressing ailments are, at present, absent, save for the provision of palliative care. Muscle stem cells, exhibiting potent self-renewal and remarkable regenerative capacity, represent a potential avenue for tackling muscular dystrophy. Due to their remarkable ability for ceaseless proliferation and diminished immunogenicity, human-induced pluripotent stem cells are viewed as a promising source for muscle stem cells. Although theoretically possible, the generation of engraftable MuSCs from hiPSCs is hampered by its relatively low efficiency and lack of consistent reproducibility. A novel transgene-free protocol for the conversion of hiPSCs into fetal MuSCs is presented, enabling the identification of the resultant cells through MYF5 positivity. The flow cytometry analysis, completed after 12 weeks of differentiation, uncovered approximately 10% of cells exhibiting a positive MYF5 phenotype. In the immunostaining analysis using Pax7, about 50 to 60 percent of the MYF5-positive cells were found to be positive. This differentiation procedure is expected to contribute significantly to both the creation of cell therapies and the future advancement of drug discovery, particularly in the context of using patient-derived induced pluripotent stem cells.

A multitude of potential uses exist for pluripotent stem cells, ranging from modeling diseases to screening drugs and developing cell-based therapies for genetic conditions, such as muscular dystrophies. Induced pluripotent stem cell technology enables the simple creation of disease-specific pluripotent stem cells for any individual patient. For the successful deployment of these applications, the targeted in vitro specialization of pluripotent stem cells into muscle cells is critical. By employing transgenes to regulate PAX7, a homogenous and expandable population of myogenic progenitors suitable for both in vitro and in vivo experimental procedures is generated. Conditional expression of PAX7 is crucial in this optimized protocol for the derivation and amplification of myogenic progenitors from pluripotent stem cells. Significantly, we present an improved technique for the terminal differentiation of myogenic progenitors into more mature myotubes, better positioned for in vitro disease modeling and drug screening analyses.

Pathological processes such as fat infiltration, fibrosis, and heterotopic ossification involve mesenchymal progenitors, which are found in the interstitial spaces of skeletal muscle. Mesenchymal progenitors, beyond their pathological contributions, are crucial for successful muscle regeneration and the maintenance of healthy muscle homeostasis. Consequently, meticulous and precise analyses of these ancestral forms are crucial for investigations into muscle disorders and well-being. Fluorescence-activated cell sorting (FACS) is used to describe a purification method for mesenchymal progenitors, identified by their expression of the specific and well-established marker, PDGFR. Cell culture, cell transplantation, and gene expression analysis are just a few of the downstream experiments that can be performed using purified cells. In addition, we describe the method for whole-mount three-dimensional imaging of mesenchymal progenitors via tissue clearing. The procedures presented herein establish a powerful system for the study of mesenchymal progenitor cells within skeletal muscle.

Efficient regeneration of adult skeletal muscle, a tissue exhibiting considerable dynamism, is supported by its stem cell system. Not only quiescent satellite cells, activated by damage or paracrine substances, but other stem cells are also implicated in adult muscle growth, either by direct or indirect actions.