Mesoangioblasts are vessel-associated stem cells initially obtained from the embryonic dorsal aorta, and, subsequently, found within the adult muscle interstitium; these cells express pericyte markers. Clinical trials for Duchenne muscular dystrophy treatment involved adult MABs, and human fetal MABs' transcriptome has been documented. Moreover, single-cell RNA sequencing studies yield novel information about adult murine muscle-associated cells (MABs) and, more generally, interstitial muscle stem cells. Advanced techniques for isolating and characterizing murine MABs, alongside fetal and adult human MABs, are discussed in this chapter.
Muscle regeneration finds its support in satellite cells, stem cells intrinsically found in skeletal muscle tissue. With increasing age and the appearance of conditions like muscular dystrophy, satellite cell populations experience a decrease. The burgeoning body of evidence underscores the essential influence of metabolic switches and mitochondrial activity on cellular destiny choices (quiescence, activation, differentiation, and self-renewal) during myogenesis. To that end, the Seahorse XF Bioanalyzer's capabilities for monitoring and characterizing metabolic profiles in living cells could offer valuable discoveries in understanding the molecular mechanisms governing stem cell dynamics during tissue regeneration and maintenance processes. In this report, we outline a procedure for determining mitochondrial respiration (oxygen consumption rate) and glycolysis (ECAR) in primary murine satellite cells, multinucleated myotubes, and C2C12 myoblasts.
Recently, mounting evidence has highlighted metabolism's role as a primary controller of stem cell functions. Satellite cells, the stem cells of skeletal muscle, play a critical role in maintaining muscle regeneration, though their regenerative potential deteriorates with age, and this is likely partly due to shifts in their metabolic processes. Employing Seahorse technology, this chapter outlines a protocol for examining the metabolic function of satellite cells, as relevant to aging mice.
Damage to myofibers prompts the activity of adult muscle stem cells for their rebuilding. Although endowed with significant power to initiate the adult myogenic program, their capacity for complete and efficient regeneration depends on environmental signals from neighboring cells. Fibroadipogenic precursors, vascular cells, and macrophages are integral to the cellular milieu that surrounds and supports muscle stem cells. Unraveling the complexity of muscle stem cell-neighboring cell communication is possible through co-culturing freshly isolated muscle cells to assess the impact of one cell type on the behavioral and developmental fate of the other. Optical biosensor To isolate primary muscle stem cells, macrophages, and fibroadipogenic precursors, a protocol utilizing Fluorescence Activated Cell Sorting (FACS) or Magnetic Cell Separation (MACS) is presented. This is followed by co-culture in a custom setup for a limited time to retain the cells' inherent in vivo properties.
In response to injury and normal wear and tear, the muscle satellite cell population is in charge of keeping muscle fibers in homeostatic balance. This population's diverse makeup and potential for self-renewal and differentiation can be altered through mutations in genes controlling these processes, or by the influence of natural processes such as aging. Extracting information about the proliferation and differentiation potential of individual cells is easily accomplished using the satellite cell colony assay. This document outlines a comprehensive protocol for isolating, plating individual cells, culturing, and assessing colonies originating from single satellite cells. Therefore, the parameters of cell survival (cloning efficacy), proliferative capability (nuclei per colony), and propensity for differentiation (ratio of myosin heavy chain-positive nuclei within the cytoplasm to all nuclei) are thus obtainable.
The physical stress on adult skeletal musculature necessitates a continuous process of maintenance and repair to ensure continued, effective functioning. Muscle hypertrophy and regeneration are both facilitated by resident muscle stem cells, satellite cells, located beneath the basal lamina of adult myofibers. The presence of activating stimuli prompts MuSCs to reproduce and create new myoblasts that specialize and fuse together to restore or augment myofibers. Along with this, teleost fish demonstrate continuous growth throughout their lifespan, requiring a continuous supply of nuclei from MuSCs to generate and expand new muscle fibers. This is unlike the determinate growth seen in most amniotes. We present a method in this chapter for the isolation, cultivation, and immuno-staining of adult zebrafish myofibers. This technique allows for the evaluation of myofiber attributes both outside the living organism and the MuSC myogenic process in a controlled environment in vitro. Repeat hepatectomy Assessing distinctions between slow and fast muscles, or exploring cellular attributes like sarcomeres and neuromuscular junctions, proves advantageous through morphometric analysis of isolated myofibers. Myogenic satellite cells (MuSCs) on isolated myofibers are visualized through Pax7 immunostaining, a technique crucial for subsequent investigation. The viable myofiber plating procedure, in addition, promotes MuSC activation and expansion, enabling downstream investigation into their proliferative and differentiative dynamics, presenting a suitable, parallel alternative to amniote models for vertebrate myogenesis research.
Skeletal muscle stem cells (MuSCs), possessing a noteworthy capacity for myogenic regeneration, have been considered a prospective treatment for various muscular disorders. Achieving better therapeutic results requires isolating human MuSCs from a suitable tissue source displaying strong myogenic differentiation characteristics. Extra eyelid tissues were subjected to the isolation of CD56+CD82+ cells, whose myogenic differentiation potential was then assessed in vitro. Primary myogenic cells, derived from the orbicularis oculi muscle and other extra eyelid tissues in humans, offer a potential avenue for human muscle stem cell-based research.
Fluorescence-activated cell sorting (FACS) is a powerful and necessary tool, proving essential for the analysis and purification of adult stem cells. Although isolating adult stem cells from immune-related tissues/organs is achievable, the separation process from solid organs is more demanding. The substantial amount of debris directly correlates to the heightened noise in the FACS profiles. find more Unfamiliar researchers, in particular, face immense difficulty in identifying muscle stem cells (also known as muscle satellite cells, MuSC), primarily due to the degradation of all myofibers—which are largely comprised of skeletal muscle tissue—during cell preparation. Our FACS protocol, a technique used for more than a decade, is described in this chapter as a method to identify and purify MuSCs.
Individuals with dementia (PwD) frequently receive prescriptions for psychotropic medications to manage non-cognitive symptoms of dementia (NCSD), but the potential risks are considerable. The Republic of Ireland (ROI)'s acute hospitals were audited nationally to evaluate baseline prescribing practices of psychotropic medications for NCSD, before the implementation of the National Clinical Guideline. The purpose of this investigation was to examine psychotropic prescribing practices, placing them in the context of international data and the constrained information from an earlier audit review.
The second round of the Irish National Audit of Dementia Care (INAD-2) yielded a pooled anonymous dataset which was subsequently analyzed. The audit, conducted in 2019, acquired retrospective data through the random selection of 30 healthcare records from each of 30 acute hospitals. To be included in the audit, participants required a clinical diagnosis of dementia, a hospital stay of at least 72 hours, and either discharge or death within the audit period. An independent self-audit of healthcare records was conducted by 87% of hospitals; however, a subsequent review of a random sample of 20% of each hospital's records was conducted by a highly trained healthcare auditor. The audit instrument was derived from the England and Wales National Audit of Dementia's audit rounds (Royal College of Psychiatrists), subsequently customized for the Irish healthcare context and national objectives.
Including 893 cases in the analysis, one hospital fell short of providing data for 30 cases, despite a more extensive audit period. A breakdown of the sample revealed 55% female and 45% male participants; the median age was 84 years, with an interquartile range of 79 to 88 years, and 89.6% of the sample were above the age of 75 years. In 52% of the examined healthcare records, the type of dementia was documented; Alzheimer's disease constituted 45% of the identified cases among those records. Upon admission, psychotropic medication was administered to 83% of PwD; during their stay, 40% received new or additional prescriptions, mainly for clinical reasons including end-of-life care and delirium treatment. Hospital-based treatment of NCSD infrequently involved the use of anticonvulsants or cognitive enhancers. A substantial amount of the study cohort, between 118-176%, received either new or elevated doses of antipsychotic medications. Simultaneously, 45-77% of the group were prescribed benzodiazepines for anxiety or neurocognitive syndrome disorders (NCSD). Concerningly, the documentation of the relationship between risks and benefits, alongside discussions with the patient or family, was deficient, and an insufficient review of the efficacy and tolerability factors was evident. The application of acetylcholinesterase inhibitors for cognitive decline in the community context, concurrently, demonstrated a seeming lack of use in sufficient amounts.
Before a specific Irish guideline was established, this audit documented the initial usage of psychotropic medication prescriptions for NCSD in Irish hospitals. Observing this trend, a significant portion of individuals with disabilities (PwD) were prescribed psychotropic medication at admission, and a large number were prescribed additional or increased doses during their hospital stay. This practice often occurred without supporting evidence of suitable prescribing and decision-making.