Name: preparation of 3d decellularized matrices from fetal mouse skeletal muscle for cell culture
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Description: Watch this Scientific Journal Video about Preparation of 3D Decellularized Matrices from Fetal Mouse Skeletal Muscle for Cell Culture at JoVE.com

This work was funded by the Association Fran&#231;aise contre les Myopathies (AFM-T&#233;l&#233;thon; contract no. 23049), the MATRIHEALTH project, and cE3c unit funding UIDB/00329/2020. We would like to thank our donor Henrique Meirelles who chose to support the MATRIHEALTH Project. This work benefitted from the infrastructures of the Faculty of Sciences Microscopy Facility, a node of the Portuguese Platform of BioImaging (reference PPBI-POCI-01-0145-FEDER-022122), and we thank Lu&#237;s Marques for his assistance with image acquisition and processing. Finally, we thank Marta Palma for technical support and our research team for their generous contributions.

All the methodologies described were approved by the Animal Welfare Committee (ORBEA) of the Faculty of Sciences, University of Lisbon, and Dire&#231;&#227;o Geral de Veterin&#225;ria (DGAV; ref. 0421/000/000/2022), and are in accordance with the European Directive 2010/63/EU.
1. Preparation of decellularization buffers and reagents
NOTE: All solutions used during the decellularization protocol should be sterilized by autoclaving and stored for up to ...

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The ECM is a complex network of macromolecules that is present in all tissues and plays a crucial role in regulating cell behavior and function2. The ECM acts as a physical scaffold for cells to attach to and provides cues that actively modulate cellular processes such as proliferation, motility, differentiation, and apoptosis. Thus, proper formation and maintenance of the ECM is essential for both development and homeostasis1.
While 2D cell cult...

The extracellular matrix (ECM) is a major constituent of tissues, representing their non-cellular component. This three-dimensional (3D) structure not only provides physical support for cells, but also plays a crucial role in the biochemical processes involved in the development of organisms1. The formation of a tissue-specific ECM occurs during development, as a result of the complex interactions between cells and their niches, influenced by various intra- and extracellular stimuli. The ECM is a highly dynamic structure that undergoes chemical and mechanical rearrangements in a temporal-spatial manner and directly impacts cell fate2. One of the most notable characteristics of the ECM is its functional diversity, as each tissue ECM displays a unique combination of molecules that provide different topologies and properties that are tailored to the cells it contains1.
ECM signaling and support are crucial for development and homeostasis, and when disrupted can lead to multiple pathological conditions3,4. One example is LAMA2-deficient congenital dystrophy (LAMA2-CMD), which is the most common form of congenital muscular dystrophy. The LAMA2 gene encodes for the laminin &#945;2 chain, which is present in laminin 211 and laminin 221, and when mutated can lead to LAMA2-CMD5. Laminin 211 is the main isoform found in the basement membrane surrounding skeletal muscle fibers. When laminin 211 is abnormal or absent, the link between the basement membrane and muscle cells is disrupted, leading to the onset of the disease6. Patients with LAMA2-CMD show a mild to severe phenotype depending on the type of mutation in the LAMA2 gene.
When the function of the laminin &#945;2 protein is affected, patients can experience severe muscle hypotonia at birth and develop chronic inflammation, fibrosis, and muscle atrophy, leading to a reduced life expectancy. To date, no targeted treatments have been developed and therapeutic approaches are limited to alleviating the symptoms of the disease7. Therefore, understanding the underlying molecular mechanisms involved in the onset of this disease is crucial for developing appropriate therapeutic strategies6,8. Previous work using the dyW mouse9, a model for LAMA2-CMD, suggests that the onset of the disease starts in utero, specifically during fetal myogenesis10. A better understanding of how the fetal myogenesis defect emerges would be a game changer in generating novel therapeutic approaches for LAMA2-CMD.
In vitro systems provide a controlled environment for studying cell-cell and cell-ECM interactions, but 2D culture models lack the complexity of native tissues. Decellularization of tissues produces tissue- and developmental stage-specific acellular ECM scaffolds that more accurately mimic the natural cell microenvironment compared to 2D models and engineered/synthetic scaffolds. Decellularized matrices (dECMs) have the potential to preserve the molecular and mechanical cues of the host tissue, making them better alternative models for understanding in vivo processes11.
There are various techniques, reagents, and conditions that can be used for decellularization12,13. In this study, a decellularization protocol for the fetal mouse heart, described by Silva et al.14,15, is adapted to fetal mouse skeletal muscle and found to retain all tested ECM components (laminin &#945;2, total laminins, fibronectin, collagen I, and collagen IV). The protocol includes three steps: cell lysis by osmotic shock (hypotonic buffer), plasma membrane dissolution and protein dissociation (0.05% sodium dodecyl sulfate [SDS]), and enzymatic destruction of DNA (DNase treatment). To our knowledge, this is the first established protocol for decellularizing mouse fetal skeletal muscle.
To use this 3D in vitro system for studying LAMA2-CMD, it is crucial to maintain the laminin &#945;2 chain after decellularization. Therefore, an optimization protocol was implemented where different detergents (SDS and Triton X-100) and concentrations (0.02%, 0.05%, 0.1%, 0.2%, and 0.5%) were tested (data not shown). The optimal choice for cell removal and preservation of the laminin &#945;2 protein was found to be 0.05% SDS. C2C12 cells, a well-established myoblast cell line16,17, were used to seed the dECMs. These cells invade the dECM, proliferate, and differentiate inside these scaffolds, synthesizing new ECM proteins. The successful production of this 3D in vitro model offers a new approach to understanding the molecular and cellular processes involved in fetal myogenesis, the onset of LAMA2-CMD, and can be extended to other muscle diseases where the communication between the ECM and skeletal muscle cells is disrupted.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >12 Well Cell Culture Plate, Flat, TC, Sterile</td>
			<td >Abdos Labware</td>
			<td >P21021</td>
			<td ></td>
		</tr>
		<tr >
			<td >4&#8242;,6-Diamidino-2-phenylindole dihydrochloride</td>
			<td >Merck</td>
			<td >D8417</td>
			<td></td>
		</tr>
		<tr >
			<td >4&#8211;20% Mini-PROTEAN TGX Precast Gel</td>
			<td >Bio-Rad</td>
			<td >4561093</td>
			<td></td>
		</tr>
		<tr >
			<td >48 Well Cell Culture Plate, Flat, TC, Sterile</td>
			<td >Abdos Labware</td>
			<td >P21023</td>
			<td></td>
		</tr>
		<tr >
			<td >96 Well Cell Culture Plate, Flat, TC, Sterile</td>
			<td >Abdos Labware</td>
			<td >P21024</td>
			<td></td>
		</tr>
		<tr >
			<td >Bovine Serum Albumin, Fraction V</td>
			<td >NZYtech</td>
			<td >MB04601</td>
			<td></td>
		</tr>
		<tr >
			<td >BX60 fluorescence microscope</td>
			<td >Olympus</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Cryostat CM1860 UV</td>
			<td >Leica </td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Dithiothreitol</td>
			<td >ThermoFisher</td>
			<td >R0862</td>
			<td></td>
		</tr>
		<tr >
			<td >DMEM high glucose w/ stable glutamine w/ sodium pyruvate</td>
			<td >Biowest</td>
			<td >L0103-500</td>
			<td></td>
		</tr>
		<tr >
			<td >DNase I</td>
			<td >PanReac AppliChem</td>
			<td >A3778</td>
			<td></td>
		</tr>
		<tr >
			<td >DNeasy Blood &#38; Tissue Kit </td>
			<td >Qiagen</td>
			<td >69506</td>
			<td></td>
		</tr>
		<tr >
			<td >Ethylenediaminetetraacetic acid (EDTA)</td>
			<td >Merck</td>
			<td >108418</td>
			<td></td>
		</tr>
		<tr >
			<td >Fetal bovine serum</td>
			<td >Biowest</td>
			<td >S1560-500</td>
			<td></td>
		</tr>
		<tr >
			<td >Fine tip transfer pipette</td>
			<td >ThermoFisher</td>
			<td >15387823</td>
			<td></td>
		</tr>
		<tr >
			<td >Goat serum</td>
			<td >Biowest</td>
			<td >S2000-100</td>
			<td></td>
		</tr>
		<tr >
			<td >Hera Guard Flow Cabinet</td>
			<td >Heraeus</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Heracell 150 CO2 Incubator</td>
			<td >Thermo Scientific</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >HiMark Pre-stained Protein Standard</td>
			<td >Invitrogen</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Horse Serum, New Zealand origin</td>
			<td >Gibco</td>
			<td >16050122</td>
			<td></td>
		</tr>
		<tr >
			<td >HRP-&#945;- Rabbit IgG</td>
			<td >abcam</td>
			<td >ab205718</td>
			<td></td>
		</tr>
		<tr >
			<td >HRP-&#945;- Rat IgG</td>
			<td >abcam</td>
			<td >ab205720</td>
			<td></td>
		</tr>
		<tr >
			<td >HRP-&#945;-Mouse IgG</td>
			<td >abcam</td>
			<td >ab205719</td>
			<td></td>
		</tr>
		<tr >
			<td >ImageJ v. 1.53t</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Methyl Green</td>
			<td >Sigma-Aldrich</td>
			<td >67060</td>
			<td></td>
		</tr>
		<tr >
			<td >MM400 Tissue Lyser</td>
			<td >Retsch</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >NanoDrop ND-1000 Spectrophotometer </td>
			<td >ThermoFisher</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Paraformaldehyde, 16% w/v aq. soln., methanol free</td>
			<td >Alfa Aesar</td>
			<td >043368-9M</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicillin-Streptomycin (100x) </td>
			<td >GRiSP</td>
			<td >GTC05.0100</td>
			<td></td>
		</tr>
		<tr >
			<td >Phalloidin Alexa 488 </td>
			<td >Thermo Fisher Sci.</td>
			<td >A12379</td>
			<td></td>
		</tr>
		<tr >
			<td >Polystyrene Petri dish 60x15mm with vents (sterile)</td>
			<td >Greiner Bio-One</td>
			<td >628161</td>
			<td></td>
		</tr>
		<tr >
			<td >Qubit dsDNA HS kit</td>
			<td >Thermo Scientific</td>
			<td >Q32851</td>
			<td></td>
		</tr>
		<tr >
			<td >Qubit&#8482; 3 Fluorometer</td>
			<td >Invitrogen</td>
			<td >15387293</td>
			<td></td>
		</tr>
		<tr >
			<td >S6E Zoom Stereo microscope</td>
			<td >Leica</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Sodium Dodecyl Sulfate</td>
			<td >Merck</td>
			<td >11667289001</td>
			<td></td>
		</tr>
		<tr >
			<td >SuperFrost&#174; Plus adhesion slides</td>
			<td >Thermo Scientific</td>
			<td >631-9483</td>
			<td></td>
		</tr>
		<tr >
			<td >SuperSignal West Pico PLUS Chemiluminescent Substrate</td>
			<td >Thermo Scientific</td>
			<td >15626144</td>
			<td></td>
		</tr>
		<tr >
			<td >TCS SPE confocal microscope</td>
			<td >Leica </td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Tris-(hidroximetil) aminometano (Tris base) &#8805;99%</td>
			<td >VWR Chemicals</td>
			<td >28811.295</td>
			<td></td>
		</tr>
		<tr >
			<td >Triton X-100</td>
			<td >Sigma-Aldrich</td>
			<td >X100-100ML</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypan Blue Solution, 0.4%</td>
			<td >Gibco</td>
			<td >15250061</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypsin-EDTA (0.05%) in DPBS (1X)</td>
			<td >GRiSP</td>
			<td >GTC02.0100</td>
			<td></td>
		</tr>
		<tr >
			<td >TWEEN 20 (50% Solution)</td>
			<td >ThermoFisher</td>
			<td >3005</td>
			<td></td>
		</tr>
		<tr >
			<td >WesternBright PVDF-CL membrane roll (0.22&#181;m)</td>
			<td >Advansta</td>
			<td >L-08024-001</td>
			<td></td>
		</tr>
		<tr >
			<td >&#945;-Collagen I</td>
			<td >abcam</td>
			<td >ab21286</td>
			<td></td>
		</tr>
		<tr >
			<td >&#945;-Collagen IV</td>
			<td >Millipore</td>
			<td >AB756P</td>
			<td></td>
		</tr>
		<tr >
			<td >&#945;-Collagen IV</td>
			<td >Santa Cruz Biotechnology</td>
			<td >sc-398655</td>
			<td></td>
		</tr>
		<tr >
			<td >&#945;-Fibronectin</td>
			<td >Sigma</td>
			<td >F-3648</td>
			<td></td>
		</tr>
		<tr >
			<td >&#945;-Laminin &#945;2 </td>
			<td >Sigma</td>
			<td >L-0663</td>
			<td></td>
		</tr>
		<tr >
			<td >&#945;-MHC</td>
			<td >D.S.H.B.</td>
			<td >MF20</td>
			<td></td>
		</tr>
		<tr >
			<td >&#945;-Mouse Alexa 488</td>
			<td >Molecular Probes</td>
			<td >A11017</td>
			<td></td>
		</tr>
		<tr >
			<td >&#945;-Mouse Alexa 568</td>
			<td >Molecular Probes</td>
			<td >A11019</td>
			<td></td>
		</tr>
		<tr >
			<td >&#945;-pan-Laminin</td>
			<td >Sigma</td>
			<td >L- 9393</td>
			<td></td>
		</tr>
		<tr >
			<td >&#945;-phospho-histone 3</td>
			<td >Merk Millipore</td>
			<td >06-570</td>
			<td></td>
		</tr>
		<tr >
			<td >&#945;-Rabbit Alexa 568</td>
			<td >Molecular Probes</td>
			<td >A21069</td>
			<td></td>
		</tr>
		<tr >
			<td >&#945;-Rabbit Alexa 488</td>
			<td >Molecular Probes</td>
			<td >A11070</td>
			<td></td>
		</tr>
		<tr >
			<td >&#945;-Rat Alexa 488</td>
			<td >Molecular Probes</td>
			<td >A11006</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of 3d decellularized matrices from fetal mouse skeletal muscle for cell culture

The extracellular matrix (ECM) plays a crucial role in providing structural support for cells and conveying signals that are important for various cellular processes. Two-dimensional (2D) cell culture models oversimplify the complex interactions between cells and the ECM, as the lack of a complete three-dimensional (3D) support can alter cell behavior, making them inadequate for understanding in vivo processes. Deficiencies in ECM composition and cell-ECM interactions are important contributors to a variety of different diseases. 
One example is LAMA2-congenital muscular dystrophy (LAMA2-CMD), where the absence or reduction of functional laminin 211 and 221 can lead to severe hypotony, detectable at or soon after birth. Previous work using a mouse model of the disease suggests that its onset occurs during fetal myogenesis. The present study aimed to develop a 3D in vitro model permitting the study of the interactions between muscle cells and the fetal muscle ECM, mimicking the native microenvironment. This protocol uses deep back muscles dissected from E18.5 mouse fetuses, treated with a hypotonic buffer, an anionic detergent, and DNase. The resultant decellularized matrices (dECMs) retained all ECM proteins tested (laminin &#945;2, total laminins, fibronectin, collagen I, and collagen IV) compared to the native tissue. 
When C2C12 myoblasts were seeded on top of these dECMs, they penetrated and colonized the dECMs, which supported their proliferation and differentiation. Furthermore, the C2C12 cells produced ECM proteins, contributing to the remodeling of their niche within the dECMs. The establishment of this in vitro platform provides a new promising approach to unravel the processes involved in the onset of LAMA2-CMD, and has the potential to be adapted to other skeletal muscle diseases where deficiencies in communication between the ECM and skeletal muscle cells contribute to disease progression.

The goal of the decellularization protocol is to produce dECMs that closely resemble the composition of native tissue. To determine the effectiveness of the decellularization process, various methods were employed, including examination of tissue morphology, measurement of DNA levels, staining for F-actin, and analysis of key ECM components using immunohistochemistry and western blotting techniques. Specifically, five major ECM components of skeletal muscle tissues were analyzed.
Throughout th...

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Preparation of 3D Decellularized Matrices from Fetal Mouse Skeletal Muscle for Cell Culture

In this work, a decellularization protocol was optimized to obtain decellularized matrices of fetal mouse skeletal muscle. C2C12 myoblasts can colonize these matrices, proliferate, and differentiate. This in vitro model can be used to study cell behavior in the context of skeletal muscle diseases such as muscular dystrophies.

The extracellular matrix (ECM) plays a crucial role in providing structural support for cells and conveying signals that are important for various ...

To our knowledge, this protocol is the first to produce decellularized matrices from fetal skeletal muscle, providing a novel approach to study myopathies that start developing in utero. This protocol generates a developmental and tissue-specific metrics that can be used to study muscle cell behavior and interactions with extracellular matrix in a controlled environment. LAMA2-CMD is a congenital muscle dystrophy that starts manifesting at birth.This model can help unravel the pattern mechanisms involved in its onset and lead to new target therapies. This protocol can be adapted to different tissues and disease models. Different complexity levels can be achieved depending on matrix and cell types, demonstrating the versatility of the system.This is a very straightforward protocol. The most challenging part is the collection and the handling of the samples. But, with practice, technical skills can easily be improved.Begin by transferring one euthanized fetus at a time to a Petri dish containing ice-cold PBS. After excising the skin and limbs, cut through the ventral side of the ribcage. Remove the sternum and underlying organs.Position the fetuses dorsal side up and remove the cervical portion of the vertebral column. Next, excise the dorsal fat deposits and the deep back muscle connective tissue. Now, anchor the ribcage using surgical forceps.With a microscalpel, carefully scrape to detach the deep back muscles from the surrounding tissue. Store the tissues in PBS in a 12-well cell culture plate at four degrees Celsius for future use. For a longer period, store the tissue in an empty microcentrifuge tube at minus 80 degrees Celsius.On the first day, use whole epaxial muscle masses. Add three milliliters of hypotonic buffer containing 1%penicillin and streptomycin to each well of a 12-well plate. Add a muscle tissue fragment to each well and incubate overnight for 18 hours with agitation.On the second day, remove the buffer with a fine-tip pipette. Now wash the samples three times with three milliliters of PBS with a one-hour agitation each time. After discarding the PBS, incubate the samples in three milliliters of 0.05%SDS detergent solution for 24 hours with agitation.On the third day, use a fine tip-pipette to remove the SDS detergent and wash the fragments thrice with three milliliters of hypotonic wash buffer with a 20-minute agitation each time. Replace the solution of the wells with two milliliters of DNase solution and incubate the tissue fragments at 37 degrees Celsius for three hours with agitation. After removing the DNase solution, wash the fragments thrice with three milliliters of PBS with a 20 minute agitation each time, leaving the last wash overnight.To a T-25 flask seeded with subconfluent C2C12 cells, add 500 microliters of trypsin and resuspend in one milliliter of complete medium. Mix 10 microliters of the suspension with 10 microliters of trypan blue dye and load into a hemocytometer to count cell number and estimate viability. In a laminar flow hood, place the decellularized matrices, or dECMs, in a Petri dish containing PBS with 1%penicillin and streptomycin.Using a microscalpel and tweezers, separate the matrices into small pieces. Transfer the fragments into the wells of a 96-well plate, placing three to four pieces per well. Add 200 microliters of prewarmed complete culture medium into each well and incubate the plate for two hours at 37 degrees Celsius with 5%carbon dioxide.Discard the medium in the well plate and add 200 microliters of complete culture medium containing 50, 000 viable C2C12 cells to each well. Incubate the well plate for two days. Transfer the dECMs with the cells to a 48 well-plate containing 400 microliters of complete culture medium.Aspirate the medium carefully every two days to prevent matrix detachment and replenish with the fresh medium until the eighth day. For differentiation, replace the complete culture medium with a differentiation medium and incubate for four days until the 12th day. The muscle tissue was reddish immediately post-isolation and turned white due to cell lysis after incubation in hypotonic buffer.SDS causes the muscles to turn transparent. After DNase treatment, a smaller, transparent dECM was obtained. DNA quantification reveals a nearly 100%decrease in the dECMs compared to the native tissue.The dECMs and native tissue displayed similar tubular staining for laminin alpha two and laminin protein. Western blot analysis of the native tissue displayed two bands for the laminin alpha two subunit, while three smaller bands were detected in the dECM. For total laminins, dECM samples showed protein fragmentation when compared to the native tissue.Fibronectin and collagen I were present in the interstitial space between cells of the native tissue and the dECMs. Similar fibronectin bands were present in both samples, indicating that it is not affected by decellularization. Fewer collagen I bands were observed in the dECMs.For collagen IV, both samples showed similar tubular staining. However, the molecular weight of the dECM bands was lower. C2C12 cells colonized and proliferated in the dECMs and fused into multi-nucleated myotubes.These expressed myosin-heavy chain protein after four days of incubation in differentiation medium. Intracellular and pericellular staining was observed for laminin alpha two chain, total laminins, and fibronectin. Decellularized fetal mouse skeletal scaffolds can be used to grow cells in co-culture systems, bringing it closer to the in vivo situation, and therefore contributes to understanding muscle disease.

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Research

JoVE Journal

Developmental Biology

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting occurs. Loss of muscle mass and function is associated with muscle fiber type atrophy, fiber type switching, defective muscle regeneration associated with dysfunction of satellite cells, muscle stem cells, and other pathophysiological processes. These changes are associated with changes in intracellular as well as local and systemic niches. In addition to most commonly used rodent models of muscle ageing, there is a need to study muscle homeostasis and wasting using human models, which due to ethical implications, consist predominantly of in vitro cultures. Despite the wide use of human Myogenic Progenitor Cells (MPCs) and primary myoblasts in myogenesis, there is limited data on using human primary myoblast and myotube cultures to study molecular mechanisms regulating different aspects of age-associated muscle wasting, aiding in the validation of mechanisms of ageing proposed in rodent muscle. The use of human MPCs, primary myoblasts and myotubes isolated from adult and aged people, provides a physiologically relevant model of molecular mechanisms of processes associated with muscle growth, atrophy and regeneration. Here we describe in detail a robust, inexpensive, reproducible and efficient protocol for the isolation and maintenance of human MPCs&#160;and their progeny&#160;&#8212; myoblasts and myotubes from human muscle samples using enzymatic digestion. Furthermore, we have determined the passage number at which primary myoblasts from adult and aged people undergo senescence in an in vitro culture. Finally, we show the ability to transfect these myoblasts and the ability to characterize their proliferative and differentiation capacity and propose their suitability for performing functional studies of molecular mechanisms of myogenesis and muscle wasting in vitro.

This protocol describes a robust, reproducible and simple method of isolation and culture of myoblast progenitor cells from the skeletal muscle of adult and aged people. The muscles used here include foot and leg muscles. This approach enables the isolation of an enriched population of primary myoblasts for functional studies.

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...