Isolation Of Muscle Stem Cells Research Articles

Characterization of Myosatellite Stem Cells from Black Bone Chicken and Effect of Different Animal Serums on Growth Performance

Isolation of muscle stem cells (myosatellite stem cells) of black bone chickens using enzymatic digestion, the pronase, and screening by low-speed centrifugation process combined with 40 μm cell strainer filtration showed that this method was able to separate chicken muscle stem cells efficiently. The results proved that the isolated cells were myosatellite stem cells expressing marker genes, c-met, Pax7, MyoD, and myogenin, by RT-PCR assay. Additionally, immunofluorescence staining with monoclonal antibodies confirmed the isolated cells were MSCs, with positive staining for canonical markers proteins: Pax7, Desmin, and actin. Evaluating the influence of fetal bovine serum (FBS), horse serum (HS), and chicken serum (CKS) supplements on MSC proliferation revealed that FBS emerged as the most effective despite its escalating cost and emerging need for alternatives. Notably, Chicken Serum (CKS) demonstrated potential as an alternative, closely following the cell proliferation rate achieved by FBS. Our findings affirm the efficacy of the devised isolation method and highlight the potential of using chicken serum in cell culture protocols. HIGHLIGHTS This research established a technique for isolating myosatellite stem cells from black bone chickens through enzymatic processes combined with gradient methods. Centrifugation aids in obtaining a purer isolation of these stem cells and allows for the evaluation of various animal serums’ effects. In cell cultures, the performance of chicken serum showed no significant difference compared to bovine serum. GRAPHICAL ABSTRACT

An Enzyme-Free Method for Isolation and Expansion of Muscle Stem Cells for Cultivated Meat Applications.

Cultivated meat, an alternative to conventional meat, holds great promise in alleviating environmental and ethical concerns. Skeletal muscle stem cell isolation is a critical phase in cultivated meat production, and efficiency is a major determinant in the final differentiated muscle cell yield. The conventional enzymatic dissociation method for cell isolation presents drawbacks, including added costs and the destruction of vital extracellular matrix components. We developed an alternative cell isolation technique, explant cell isolation, to isolate muscle stem cells from muscle tissue. The present protocol yields myogenic cell populations, mainly composed of skeletal muscle stem cells without the use of enzymes, and through a simplified process. Overall, the explant method allows for propagation of cells in their natural environment, preserving intricate cell-cell and cell-matrix interactions, resulting in both economic efficiency and consistent generation of high-quality cells.

Muscle stem cells contribute to long-term tissue repletion following surgical sepsis.

Over the past decade, advances in sepsis identification and management have resulted in decreased sepsis mortality. This increase in survivorship has highlighted a new clinical obstacle: chronic critical illness (CCI), for which there are no effective treatment options. Up to half of sepsis survivors suffer from CCI, which can include multi-organ dysfunction, chronic inflammation, muscle wasting, physical and mental disabilities, and enhanced frailty. These symptoms prevent survivors from returning to regular day-to-day activities and are directly associated with poor quality of life. Mice were subjected to cecal ligation and puncture (CLP) with daily chronic stress (DCS) as an in vivo model to study sepsis late-effects/sequelae on skeletal muscle components. Longitudinal monitoring was performed via magnetic resonance imaging, skeletal muscle and/or muscle stem cell (MuSCs) assays (e.g., post-necropsy wet muscle weights, minimum Feret diameter measurements, in vitro MuSC proliferation and differentiation, number of regenerating myofibres and numbers of Pax7-positive nuclei per myofibre), post-sepsis whole muscle metabolomics and MuSC isolation and high-content transcriptional profiling. We report several findings supporting the hypothesis that MuSCs/muscle regeneration are critically involved in post-sepsis muscle recovery. First, we show that genetic ablation of muscle stem cells (MuSCs) impairs post-sepsis muscle recovery (maintenance of 5-8% average lean mass loss compared with controls). Second, we observe impaired MuSCs expansion capacity and morphological defects at 26days post-sepsis compared with control MuSCs (P<0.001). Third, when subjected to an experimental muscle injury, sepsis-recovered mice exhibited evidence of impaired muscle regeneration compared with non-septic mice receiving the same muscle injury (CLP/DCS injured mean minimum Feret is 92.1% of control injured, P<0.01). Fourth, we performed a longitudinal RNA sequencing study on MuSCs isolated from post-sepsis mice and found clear transcriptional differences in all post-sepsis samples compared with controls. At Day 28, CLP/DCS mice satellite cells have multiple altered metabolic pathways, such as oxidative phosphorylation, mitochondrial dysfunction, sirtuin signalling and oestrogen receptor signalling, compared with controls (P<0.001). Our data show that MuSCs and muscle regeneration are required for effective post-sepsis muscle recovery and that sepsis triggers morphological, functional, and transcriptional changes in MuSCs. Moving forward, we strive to leverage a more complete understanding of post-sepsis MuSC/regenerative defects to identify and test novel therapies that promote muscle recovery and improve quality of life in sepsis survivors.

Yapay et üretimi ve gelecek vizyonu

Artificial meat shows great promise as a method for use in future food production. It is predicted that traditional meat will be insufficient with the increasing human population. In addition, artificial meat has many advantages in terms of human health, such as being sustainable for the environment, controlled fat content, and absence of antibiotics and hormones compared to traditional meat. Artificial meat, also known as cultured meat, is produced through in vitro myogenesis, which includes muscle tissue-based protein products, stem cell culture, and differentiation, and mature muscle cell processing for flavor and texture. Artificial meat production consists of a sequential process; firstly muscle sampling for stem cell collection and followed by muscle tissue dissociation and muscle stem cell isolation, primary cell culture, high cell culture, and ending with muscle differentiation and maturation. A deep understanding of the process by considering its pros and cons will help not only artificial meat production but also the food industry in business sectors seeking new biomaterials. By explaining the methods utilized for artificial meat production, this study is created to prepare for the new era of cellular agriculture as well as for application in academia and industry.

Muscle stem cell isolation and in vitro culture for meat production: A methodological review.

Cultured muscle tissue-based protein products, also known as cultured meat, are produced through in vitro myogenesis involving muscle stem cell culture and differentiation, and mature muscle cell processing for flavor and texture. This review focuses on the in vitro myogenesis for cultured meat production. The muscle stem cell-based in vitro muscle tissue production consists of a sequential process: (1) muscle sampling for stem cell collection, (2) muscle tissue dissociation and muscle stem cell isolation, (3) primary cell culture, (4) upscaled cell culture, (5) muscle differentiation and maturation, and (6) muscle tissue harvest. Although muscle stem cell research is a well-established field, the majority of these steps remain to be underoptimized to enable the in vitro creation of edible muscle-derived meat products. The profound understanding of the process would help not only cultured meat production but also business sectors that have been seeking new biomaterials for the food industry. In this review, we discuss comprehensively and in detail each step of cutting-edge methods for cultured meat production. This would be meaningful for both academia and industry to prepare for the new era of cellular agriculture.

A Modified Pre-plating Method for High-Yield and High-Purity Muscle Stem Cell Isolation From Human/Mouse Skeletal Muscle Tissues.

Primary culture of skeletal muscle stem cells (MuSCs) is indispensable to study the dynamics of muscle regeneration and homeostasis. Here we describe the modified pre-plating method for isolating MuSCs in culture with greatly improved purity, yield, and procedure time. The protocol is based on the distinct adhesion characteristics of MuSCs. We reduced the procedure time to 2.5 days to obtain highly purified MuSCs through a newly employed re-plating step, which repeats incubation and cell-suspension. The re-plating step efficiently traps remaining fibroblastic cells, but not MuSCs, on a collagen-coated dish. Additionally, we confirmed that MuSCs can be isolated from small amounts of human/mouse muscle tissues, enabling us to perform experiments with amount-limited specimens. Thus, our method can be performed with basic laboratory equipment suitable for most facilities and without sophisticated MuSC handling techniques.

Isolation of satellite cells and transplantation into mice for lineage tracing in muscle.

Limited methods exist to assay the direct effects of therapeutic intervention on muscle stem cell fate, proliferation or differentiation in an in vivo context. Here we provide an optimized protocol for muscle stem cell isolation and transplantation into mice to deconvolute heterogeneity within isolated stem cell populations. Viable and pure cell populations are isolated within 2 h and can then be used for therapeutic intervention or transplantation to uncover the repopulating and differentiation potential in mice, a physiologically relevant in vivo context. Effects can be assessed 9 d after transplantation. This methodology analyzes cell and sort purity prior to transplantation to improve reproducibility and outlines novel blocking steps to improve tissue staining and analysis. Experience with surgical procedures in mice is recommended before attempting this protocol. Our system is widely applicable for exploring stem cell dynamics within muscle and has already been used to study heterogeneity within muscle stem cell populations and efficacy of therapeutic intervention on isolated stem cell populations.

Isolation of muscle stem cells from rat skeletal muscles.

Muscle stem cells (MuSCs) are involved in homeostatic maintenance of skeletal muscle and play a central role in muscle regeneration in response to injury. Thus, understanding MuSC autonomous properties is of fundamental importance for studies of muscle degenerative diseases and muscle plasticity. Rat, as an animal model, has been widely used in the skeletal muscle field, however rat MuSC isolation through fluorescence-activated cell sorting has never been described. This work validates a protocol for effective MuSC isolation from rat skeletal muscles. Tibialis anterior was harvested from female rats and digested for isolation of MuSCs. Three protocols, employing different cell surface markers (CD106, CD56, and CD29), were compared for their ability to isolate a highly enriched MuSC population. Cells isolated using only CD106 as a positive marker showed high expression of Pax7, ability to progress through myogenic lineage while in culture, and complete differentiation in serum-deprived conditions. The protocol was further validated in gastrocnemius, diaphragm, and the individual components of the pelvic floor muscle complex (coccygeus, iliocaudalis, and pubocaudalis), proving to be reproducible. CD106 is an efficient marker for reliable isolation of MuSCs from a variety of rat skeletal muscles.

Technical advantage of recombinant collagenase for isolation of muscle stem cells.

BackgroundMuscle satellite cells are resident skeletal muscle stem cells responsible for muscle regeneration. Isolation of satellite cells is a critical process for clinical application such as drug screening and cell transplantation. Fluorescence-activated cell sorting (FACS) enables the direct isolation of satellite cells from muscle tissue. During the process used to isolate satellite cells from skeletal muscle, enzymatic digestion is the first step. Therefore, the evaluation and standardization of enzymes is important not only for reproducibility of cellular yield and viability, but also for traceability of material used in protocols.MethodsThe comparison of muscle digestion was performed either by a mixture of recombinant collagenase G (ColG) and collagenase H (ColH) or by a conventional collagenase II. The degree of cell damage and surface antigen expression upon collagenase treatment were analyzed by FACS. To investigate whether satellite cells isolated using recombinant collagenase can regenerate injured muscle, satellite cells were cultured, transplanted into injured muscles, and analyzed by immunostaining.ResultsWe show that ColG and ColH were efficient to isolate satellite cells from mouse skeletal muscle tissue. Digestion with a combination of ColG and ColH enriched satellite cells with intact surface antigens such as α7 and β1 integrins. Furthermore, satellite cells isolated using ColG and ColH dramatically proliferated and remained undifferentiated in vitro. When transplanted, satellite cells isolated using ColG and ColH enhanced the therapeutic efficacy in vivo.ConclusionsOur results provide an efficient method of satellite cell preparation using recombinant collagenases with a high cell yield, viability of cells, and regeneration potency to fit the biological raw material criteria.

Isolation of Muscle Stem Cells from Mouse Skeletal Muscle.

Isolation of muscle stem cells from skeletal muscle is a critical step for the study of skeletal myogenesis and regeneration. Although stem cell isolation has been performed for decades, the emergence of flow cytometry with defined cell surface markers, or transgenic mouse models, has allowed the efficient isolation of highly enriched stem cell populations. Here, we describe the isolation of mouse muscle stem cells using two different combinations of enzyme treatments allowing the release of mononucleated muscle stem cells from their niche. Mouse muscle stem cells can be further isolated as a highly enriched population by flow cytometry using fluorescent reporters or cell surface markers. We will present advantages and drawbacks of these different approaches.

Muscle-derived stem cells in tissue engineering: defining cell properties suitable for construct design.

The terms construct or tissue equivalent refer to neotissue produced by tissue engineering techniques. The elements forming the construct are scaffolds on which cells are "recreated" to form an engineered-tissue sensitive to certain cell signals. The ability of the cells to expand and differentiate on the scaffold is determined by properties such as fixation, adhesion, proliferation and migration. Among the cell types that seem to be most promising for designing constructs are tissue-residing, or adult, stem cells, which show two main features: a capacity to differentiate into many cell lineages and the power of self-renewal. These features make them good candidates for cell replacement therapies. Here, we report the identification, isolation and culture of muscle stem cells aimed at establishing the ideal culture in terms of defining when the cultured cell population would show optimal characteristics for transfer to the scaffold to obtain a particular construct. Stem cells harvested from the dorsal muscle of white New Zealand rabbits were cultured in vitro and characterized 5 to 14 days after the start of culture. Fibroblasts obtained from the same experimental animal served as controls. The stem cells were examined by light and scanning electron microscopy. For stem cell identification, we used the antibodies anti-m-cadherin, anti-CD34 and anti-Myf-5. The markers of muscle differentiation used were: anti-vimentin, anti-alpha-actin, anti-desmin and anti-myosin. The expression profiles of the different markers of muscle differentiation and TGFbeta1 in the cell cultures were confirmed by Western blotting. Proliferation rates were determined by monitoring tritiated thymidine incorporation. The thymidine incorporation rate was substantially higher for the population of undifferentiated cells than for control fibroblasts obtained from the same animal. During the first five days of culture, most cells were negative for all the markers examined, with the exception of m-cadherin, CD34 and Myf-5, although discrete signs of vimentin expression started to emerge. After 14 days of culture, the adult stem cells showed vimentin (94.2%) and desmin (33.8%) expression yet scarce labeling for myosin (16.2%) and alpha-actin (8.3%). Control fibroblasts showed intense labeling for vimentin (99.3%) and alpha-actin (62.2%), while less than 2% of the population expressed myosin (0.9%) and desmin (1.6%). After two weeks of culture, muscle-derived stem cells show good proliferative and adhesion properties as they initiate differentiation. These conditions seem ideal for obtaining the desired construct.

Transfection of MCF-7 Carcinoma Cells with Human Integrin α7 cDNA Promotes Adhesion to Laminin

The laminin-binding α7β1 integrin receptor is highly expressed by skeletal and cardiac muscles, and has been suggested to be a crucial molecule during myogenic cell migration and differentiation. Absence of integrin α7 subunit contributes to a form of muscular dystrophy in integrin α7 null mice, whereas specific mutations in the α7 gene are associated in humans with congenital myopathy. To examine in more detail the potential role of integrin α7 in human-related muscular disorders, we cloned α7 cDNA by RT-PCR from human skeletal muscle mRNA and then expressed the full-length human integrin α7 cDNA by transfection in several cell lines including MCF-7, COS-7, and NIH3T3 cells. The isolated cDNA corresponds to the human α7X2B alternative splice form. Expression of human α7 was further confirmed by transfection of chimeric human/mouse α7 cDNA constructs. To demonstrate the functionality of expressed human α7, adhesion experiments with transfected MCF-7 cells have confirmed the specific binding of human α7 to laminin. In addition, mouse polyclonal and monoclonal antibodies were generated against the extracellular domain of human α7 and used to analyze by flow cytometry MCF-7 and NIH3T3 cells transfected with the full-length of human α7 cDNA. These results show for the first time the exogenous expression of functional full-length human α7 cDNA, as well as the development of monoclonal antibodies against the human α7 extracellular domain. Antibodies developed will be useful for further analysis of human disorders involving α7 dysfunction and facilitate isolation of muscle stem cells (satellite cells) and thereby expand the opportunities for genetically modified transplantation treatment of human disease.