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 ...

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