Name: performing human skeletal muscle xenografts in immunodeficient mice
Uploaded: 2019-09-16T14:30:00
Description: Watch this Scientific Journal Video about Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice at JoVE.com

This work was supported by The Myositis Association and the Peter Buck Foundation. We would like to thank Dr. Yuanfan Zhang for sharing her expertise and training in the xenograft surgical technique.

All use of research specimens from human subjects was approved by the Johns Hopkins Institutional Review Board (IRB) to protect the rights and welfare of the participants. All animal experiments were approved by the Johns Hopkins University Institutional Animal Care and Use Committee (IACUC) in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Male NOD-Rag1null IL2r&#947;null (NRG) host mice (8-12 weeks old) ...

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Patient-derived xenografts are an innovative way to model muscle disease and carry out preclinical studies. The method described here to create skeletal muscle xenografts is rapid, straightforward, and reproducible. Unilateral surgeries can be performed in 15 to 25 minutes, or bilaterally in 30 to 40 minutes. Bilateral xenografts can provide additional experimental flexibility. For instance, researchers can perform localized treatment of one xenograft, with the other left as a control. The NRG mice are resistant to surgi...

It has been reported that only 13.8% of all drug development programs undergoing clinical trials are successful and lead to approved therapies1. While this success rate is higher than the 10.4% previously reported2, there is still significant room for improvement. One approach to increase the success rate of clinical trials is to improve laboratory models used in preclinical research. The Food and Drug Administration (FDA) requires animal studies to show treatment efficacy and assess toxicity prior to Phase 1 clinical trials. However, there is often limited concordance in treatment outcomes between animal studies and clinical trials3. In addition, the need for preclinical animal studies can be an insurmountable barrier for therapeutic development in diseases that lack an accepted animal model, which is often the case for rare or sporadic diseases.
One way to model human disease is by transplanting human tissue into immunodeficient mice to generate xenografts. There are three key advantages to xenograft models: First, they can recapitulate the complex genetic and epigenetic abnormalities that exist in human disease that may never be reproducible in other animal models. Second, xenografts can be used to model rare or sporadic diseases if patient samples are available. Third, xenografts model the disease within a complete in vivo system. For these reasons, we hypothesize that treatment efficacy results in xenograft models are more likely to translate to trials in patients. Human tumor xenografts have already been successfully utilized to develop treatments for common cancers, including multiple myeloma, as well as personalized therapies for individual patients4,5,6,7.
Recently, xenografts have been used to develop a model of human muscle disease8. In this model, human muscle biopsy specimens are transplanted into the hindlimbs of immunodeficient NRG mice to form xenografts. The transplanted human myofibers die, but human muscle stem cells present in the xenograft subsequently expand and differentiate into new human myofibers which repopulate the engrafted human basal lamina. Therefore, the regenerated myofibers in these xenografts are entirely human and are spontaneously revascularized and innervated by the mouse host. Importantly, fascioscapulohumeral muscular dystrophy (FSHD) patient muscle tissue transplanted into mice recapitulates key features of the human disease, namely expression of the DUX4 transcription factor8. FSHD is caused by overexpression of DUX4, which is epigenetically silenced in normal muscle tissue9,10. In the FSHD xenograft model, treatment with a DUX4-specific morpholino has been shown to successfully repress DUX4 expression and function, and may be a potential therapeutic option for FSHD patients11. These results demonstrate that human muscle xenografts are a new approach to model human muscle disease and test potential therapies in mice. Here, we describe in detail the surgical method for creating human skeletal muscle xenografts in immunodeficient mice.

<table>
	<tbody>
		<tr>
			<td>100 mm x 15 mm Petri dish</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Methylbutane</td>
			<td>Fisher</td>
			<td>O3551-4</td>
			<td></td>
		</tr>
		<tr>
			<td>20 x 30 mm micro cover glass</td>
			<td>VWR</td>
			<td>48393-151</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal Weighing Scale</td>
			<td>Kent Scientific</td>
			<td>SCL- 1015</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic Solution</td>
			<td>Corning, Cellgro</td>
			<td>30-004-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>AutoClip System</td>
			<td>F.S.T</td>
			<td>12020-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Castroviejo Needle Holder</td>
			<td>F.S.T</td>
			<td>12565-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Chick embryo extract</td>
			<td>Accurate</td>
			<td>CE650TL</td>
			<td></td>
		</tr>
		<tr>
			<td>CM1860 UV cryostat</td>
			<td>Leica Biosystems</td>
			<td>CM1860UV</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplin staining jar</td>
			<td>Thermo Scientific</td>
			<td>19-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Pins</td>
			<td>Fisher Scientific</td>
			<td>S13976</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry Ice - pellet</td>
			<td>Fisher Scientific</td>
			<td>NC9584462</td>
			<td></td>
		</tr>
		<tr>
			<td>Embryonic Myosin antibody</td>
			<td>DSHB</td>
			<td>F1.652</td>
			<td>recommended concentration 1:10</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>459836</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30071.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber-Lite MI-150</td>
			<td>Dolan-Jenner</td>
			<td>Mi-150</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>F.S.T</td>
			<td>11295-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG1, Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-21121</td>
			<td>recommended concentration 1:500</td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG2b, AlexaFluor 594</td>
			<td>Invitrogen</td>
			<td>A-21145</td>
			<td>recommended concentration 1:500</td>
		</tr>
		<tr>
			<td>Gum tragacanth</td>
			<td>Sigma</td>
			<td>G1128</td>
			<td></td>
		</tr>
		<tr>
			<td>Hams F-10 Medium</td>
			<td>Corning</td>
			<td>10-070-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Histoacryl Blue Topical Skin Adhesive</td>
			<td>Tissue seal</td>
			<td>TS1050044FP</td>
			<td></td>
		</tr>
		<tr>
			<td>Human specific lamin A/C antibody</td>
			<td>Abcam</td>
			<td>ab40567</td>
			<td>recommended concentration 1:50-1:100</td>
		</tr>
		<tr>
			<td>Human specific spectrin antibody</td>
			<td>Leica Biosystems</td>
			<td>NCLSPEC1</td>
			<td>recommended concentration 1:20-1:100</td>
		</tr>
		<tr>
			<td>Induction Chamber</td>
			<td>VetEquip</td>
			<td>941444</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Forceps</td>
			<td>F.S.T</td>
			<td>11066-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Irradiated Global 2018 (Uniprim 4100 ppm)</td>
			<td>Envigo</td>
			<td>TD.06596</td>
			<td>Antibiotic rodent diet to protect again respiratory infections</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>MWI Veterinary Supply</td>
			<td>502017</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark</td>
			<td>34155</td>
			<td>surgical wipes</td>
		</tr>
		<tr>
			<td>Mapleson E Breathing Circuit</td>
			<td>VetEquip</td>
			<td>921412</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A412</td>
			<td></td>
		</tr>
		<tr>
			<td>Mobile Anesthesia Machine</td>
			<td>VetEquip</td>
			<td>901805</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse on Mouse Basic Kit</td>
			<td>Vector Laboratories</td>
			<td>BMK-2202</td>
			<td>mouse IgG blocking reagent</td>
		</tr>
		<tr>
			<td>Nail Polish</td>
			<td>Electron Microscopy Sciences</td>
			<td>72180</td>
			<td></td>
		</tr>
		<tr>
			<td>NAIR Hair remover lotion/oil</td>
			<td>Fisher Scientific</td>
			<td>NC0132811</td>
			<td></td>
		</tr>
		<tr>
			<td>NOD-Rag1null IL2rg null (NRG) mice</td>
			<td>The Jackson Laboratory</td>
			<td>007799</td>
			<td>2 to 3 months old</td>
		</tr>
		<tr>
			<td>O.C.T. Compound</td>
			<td>Fisher Scientific</td>
			<td>23-730-571</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen</td>
			<td>Airgas</td>
			<td>OX USPEA</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (phosphate buffered saline) buffer</td>
			<td>Fisher Scientific</td>
			<td>4870500</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone Iodine Prep Solution</td>
			<td>Dynarex</td>
			<td>1415</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong&#8482; Gold Antifade Mountant</td>
			<td>Fisher Scientific</td>
			<td>P10144 (no DAPI); P36935 (with DAPI)</td>
			<td></td>
		</tr>
		<tr>
			<td>Puralube Ophthalmic Ointment</td>
			<td>Dechra</td>
			<td>17033-211-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Rimadyl (carprofen) injectable</td>
			<td>Patterson Veterinary</td>
			<td>10000319</td>
			<td>surgical analgesic, administered subcutaneously at a dose of 5mg/kg</td>
		</tr>
		<tr>
			<td>Scalpel Blades - #11</td>
			<td>F.S.T</td>
			<td>10011-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Handle - #3</td>
			<td>F.S.T</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Accu-scope</td>
			<td>3075</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture, Synthetic, Non-Absorbable, 30 inches long, CV-11 needle</td>
			<td>Covidien</td>
			<td>VP-706-X</td>
			<td></td>
		</tr>
		<tr>
			<td>1ml Syringe (26 gauge, 3/8 inch needle)</td>
			<td>BD Biosciences</td>
			<td>329412</td>
			<td></td>
		</tr>
		<tr>
			<td>Trimmer</td>
			<td>Kent Scientific</td>
			<td>CL9990-KIT</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors, 8.0 mm cutting edge</td>
			<td>F.S.T</td>
			<td>15009-08</td>
			<td></td>
		</tr>
		<tr>
			<td>VaporGaurd Activated Charcoal Filter</td>
			<td>VetEquip</td>
			<td>931401</td>
			<td></td>
		</tr>
		<tr>
			<td>Wound clips, 9 mm</td>
			<td>F.S.T</td>
			<td>12022-09</td>
			<td></td>
		</tr>
	</tbody>
</table>

performing human skeletal muscle xenografts in immunodeficient mice

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this failure is the use of inadequate laboratory models. It is challenging to model complex human diseases in traditional laboratory organisms, but this issue can be circumvented through the study of human xenografts. The surgical method we describe here allows for the creation of human skeletal muscle xenografts, which can be used to model muscle disease and to carry out preclinical therapeutic testing. Under an Institutional Review Board (IRB)-approved protocol, skeletal muscle specimens are acquired from patients and then transplanted into NOD-Rag1null IL2r&gamma;null (NRG) host mice. These mice are ideal hosts for transplantation studies due to their inability to make mature lymphocytes and are thus unable to develop cell-mediated and humoral adaptive immune responses. Host mice are anaesthetized with isoflurane, and the mouse tibialis anterior and extensor digitorum longus muscles are removed. A piece of human muscle is then placed in the empty tibial compartment and sutured to the proximal and distal tendons of the peroneus longus muscle. The xenografted muscle is spontaneously vascularized and innervated by the mouse host, resulting in robustly regenerated human muscle that can serve as a model for preclinical studies.

As demonstrated by Yuanfan Zhang et al., this surgical protocol is a straightforward method to produce human skeletal muscle xenografts8. Regenerated xenografts become spontaneously innervated and display functional contractility. In addition, muscle xenografted from FSHD patients recapitulates changes in gene expression observed in FSHD patients8.
In our experience, approximately 7 out of 8 xenografts performed from control patient specimens wil...

Watch this Scientific Journal Video about Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice at JoVE.com

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice

Complex human diseases can be challenging to model in traditional laboratory model systems. Here, we describe a surgical approach to model human muscle disease through the transplantation of human skeletal muscle biopsies into immunodeficient mice.

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this ...

This protocol is significant because it can be used to create human skeletal muscle xenografts, which can be used to model muscle disease and to carry out preclinical therapeutic testing. This xenograft model allows researchers to study human muscle in vivo to better understand human muscle cell biology and to develop novel models for rare or acquired muscle diseases. After obtaining a human muscle biopsy, place the specimen in a 100-by-15-millimeter Petri dish containing muscle medium, and use a stereo microscope and surgical scissors to remove any remaining fascia or fatty tissue.Dissect the muscle biopsy into approximately seven-by-three-by-three-millimeter pieces, taking care that the fibers are arranged longitudinally within the specimen. Next, transfer the Petri dish containing the dissected muscle on ice, and place synthetic, non-absorbable sutures in a 100-by-15-millimeter Petri dish of 70%ethanol. After confirming a lack of response to toe pinch in an eight-to 12-week-old, anesthetized NOD-Rag-gamma mouse, apply ointment to the immunodeficient animal's eyes, and use a trimmer to remove the hair overlying the tibialis anterior from the ankle to the knee.Then, apply depilatory cream to the exposed skin for one minute before disinfecting the surgical site with povidone-iodine solution and 70%ethanol. To graft the human tissue, first tape down the mouse leg, and make a straight incision over the tibialis anterior originating at the distal tendons and terminating below the knee. Use blunt dissection to separate the skin from the muscle tissue, and use scissors to make a less-than-0.5-millimeter incision through the epimysium of the tibialis anterior muscle, starting at the tendon and ending at the knee.After cutting the distal tendon of the tibialis anterior, grasp the tendon with iris forceps, and pull the muscle up toward the knee. Next, cut the distal tendon of the extensor digitorum longus, and pull the extensor digitorum longus up toward the knee. Once the proximal tendon of the peroneus longus muscle is visible, remove the extensor digitorum longus and the tibialis anterior with the scissors.Use a surgical wipe wet with PBS to apply slight pressure until hemostasis is achieved, and thread a suture through the proximal peroneus longus tendon, leaving an approximately 1.5-inch piece of thread on either side of the tendon. Make the first half of a two-hand surgical square knot without tightening to form a circle, and place a xenograft into the circle, tightening the loop to secure the graft tissue. Complete the other half of the square knot to suture the xenograft to the proximal tendon of the peroneus longus, and thread the suture through the distal peroneus longus tendon to repeat the square knot technique at the distal tendon.When both ends of the graft have been secured, pull the skin over the xenografted muscle, seal the incision with surgical glue, and place two to three surgical staples over the glue. Then, place the mouse in a clean cage on a heated pad with monitoring until full recumbency. Four to six months after the surgery, place a covered beaker containing 200 milliliters of 2-methylbutane in a box of dry ice for at least 30 minutes before the xenograft harvest.When the 2-methylbutane has cooled, confirm a lack of response to toe pinch, and remove the hair overlying the tibialis anterior from the ankle to the knee with a trimmer and depilatory cream. The sutures holding the xenograft in place should be observed through the skin. Secure the leg with tape, and use scissors and iris forceps to open the skin over the xenograft until both sutures are uncovered.Use a scalpel to cut between the xenograft and the tibia to free one side of the xenograft before cutting between the peroneus longus muscle and the gastrocnemius muscle. Cut below the distal suture and through the distal tendon of the peroneus longus, and use the iris forceps to grasp and deflect the suture toward the knee while using scissors to cut the xenograft away from the underlying muscle tissue. Then, use scissors to cut above the proximal suture to remove the xenograft and peroneus longus, and place the harvested specimen on a small piece of cardboard.Pin the tissue as close to the sutures as possible while gently stretching the muscle to ensure that the fiber orientation will be maintained during the snap-freezing process. When the pins are securely in place, slide the muscle up the pins so the tissue rests just above the cardboard, and snap freeze the xenograft in the pre-cooled 2-methylbutane for minus 80-degree Celsius storage. A successful xenograft demonstrates a robust regeneration of human myofibers as identified with human-specific antibodies.Positive embryonic myosin staining within a proportion of myofibers indicates that the regeneration process is still ongoing. In contrast, poor surgical technique or an inadequate specimen may lead to a poor regeneration of the muscle fibers. Xenografts performed from a patient diagnosed with an idiopathic inflammatory myopathy exhibit moderate numbers of regenerated human myofibers at four-and six-month collections, and embryonic myosin staining persists at six months.Inflammatory cells are also present within the xenograft, as observed by H and E staining. Individual myofiber sizes are similar between four and six months in idiopathic inflammatory myopathy xenografts. Rare fibers showing a cross-sectional area greater than 3, 500 micrometers squared are observed within the xenografts but not in the idiopathic inflammatory myopathy biopsies, indicating that some of the myofibers in the xenografts can regenerate to a cross-sectional area similar in size to healthy myofibers.The most important parts of this procedure are identifying all of the tendons in the ankle after the initial incision is made and successfully cutting through the epimysium. The functional competency of the xenografts can be assessed by enzymatically isolating single myofibers and testing calcium transients or by performing evoked forced measurements following electrical stimulation.

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The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

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 ...

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 ...

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

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 ...

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

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 ...

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 ...