We would like to acknowledge Andy Johnson and Justin Wong, UBC flow core, for their expertise and help in optimizing the FACS protocol, as well as the UBC Biomedical Research Center animal facility staff for spiny mouse care. Figure 1 has been made using Biorender. Figure 2 has been made using FlowJo software.

All animal maintenance and experimental procedures were conducted in accordance with the approval of the University of British Columbia Animal Care Committee and the regulations at the University of British Columbia. Animals were housed in an enclosed pathogen-free facility under standard conditions (12:12 light-dark cycle, 21-23 &#176;C, and 40%-60% humidity level) and provided a protein-rich mouse diet and water ad libitum. Adult (4 to 6 months old, 50-60 g) female and male Acomys dimidiatus mice were used for this study. The details of all the reagents and equipment used are listed in the Table of Materials.
1. Solution and buffer preparation
<ol>
	<li>Prepare 1x Phosphate Buffer Saline (PBS).</li>
	<li>Prepare PBS-EDTA by mixing 4 mL of 0.5 M EDTA (2 mM) and 996 mL of 1x PBS, pH = 7.4.</li>
	<li>Prepare 250 mM CaCl2 by adding 2.7745 g of CaCl2 to 100 mL of nuclease-free water.</li>
	<li>Prepare digestion buffer: Mix 50 &#181;g/mL liberase, 2.5 mM CaCl2, and 5% fetal bovine serum (FBS) in DMEM/F12.</li>
	<li>Prepare FACS buffer by mixing 488 mL of 1x PBS, 2 mL of 0.5M EDTA, and 10 mL of FBS.</li>
	<li>Prepare Viability buffer by mixing FACS buffer with propidium iodide (PI; 1 &#181;g/mL).</li>
	<li>Prepare Basic media by mixing DMEM/F12 with 5% FBS, and 1% (v/v) pen/strep.</li>
	<li>Prepare Proliferation media by mixing DMEM/F12 with 10% FBS, 1% (v/v) pen/strep and 2.5 ng/mL bFGF.</li>
	<li>Prepare Fibrogenic media by mixing Basic media and 10 ng/mL rh-TGF&#946;1,</li>
	<li>Prepare Adipogenic media (1x) by mixing DMEM/F12 with 1% pen/strep and a commercially available 10x mouse adipogenic differentiation supplement.</li>
	<li>Prepare 4% PFA by adding 4% Paraformaldehyde (w/v) to 1x PBS.</li>
	<li>Prepare 0.3% PBS-Triton by mixing 498.5 mL of 1x PBS and 1.5 mL of TritonX-100.</li>
	<li>Prepare Donkey blocking buffer by mixing 5% (v/v) Donkey serum, 1% BSA (w/v), and 0.3% PBS-Triton.</li>
	<li>Prepare Donkey + MOM blocking buffer by mixing 2.5 mL of Donkey blocking buffer and 2 drops of MOM block.</li>
	<li>Prepare DAPI staining solution by mixing 600 nM of 4&#8242;,6-diamidino-2-phenylindole (DAPI) in 1x PBS.</li>
</ol>
2. Tissue collection
<ol>
	<li>Euthanize the animal following Institutional Animal Care and Use Committee guidelines. For Spiny mice, isoflurane followed by CO2 (20% of the volume of the cage/min) and spinal dislocation to ensure death is recommended.</li>
	<li>Place the mouse on the dissection stage in the supine position, and thoroughly spray with 70% ethanol to prevent contamination with mouse fur.</li>
	<li>Make a 2-3 cm vertical incision at the ventral midline and expose the rib cage and abdominal muscle. Cut through the muscle at the xiphoid process with tissue scissors and expose the diaphragm. Cut the diaphragm and rib cage on both sides. Use a hemostat to clamp and peel back the cut ribs.</li>
	<li>Nick the right atrium with tissue scissors, place a 23 G needle attached to a 20 mL syringe in the apex of the heart, and perfuse it by slowly injecting 20 mL of 2 mM PBS-EDTA.</li>
	<li>Excise the heart, rinse in cold 1x PBS in a 60 mm Petri dish on ice, remove atria, and clean blood as much as possible.</li>
	<li>Remove the skin above the knee joint. Use fine forceps to remove the fascia and isolate the quadricep muscle with scissors by using the femur as a guide. Place in a separate Petri dish with cold 1x PBS.</li>
	<li>Repeat step 2.6 for the other leg.</li>
</ol>
3. Tissue digestion
<ol>
	<li>Add 2 mL of digestion buffer to a 5 mL transport vial for the heart, and 4 mL of digestion buffer to a 15 mL centrifuge tube for two quadricep muscles.</li>
	<li>Mince the tissue with tissue scissors on the lid of the Petri dish into smaller than 1 mm3 pieces.</li>
	<li>Add minced tissue to their respective digestion tubes. Vortex for 2 s at low speed to mix.</li>
	<li>Incubate at 37 &#176;C with gentle rotation and shaking.</li>
	<li>After 20 min, take the tubes off the rotator and allow the tissue pieces to settle. Take off the supernatant with a P1000 pipette into 20 mL of ice-cold FACS buffer in 50 mL centrifuge tubes.</li>
	<li>Top up the digestion tubes with 2 mL and 4 mL of digestion buffer for heart and muscle digest respectively, and place them back at 37 &#176;C with gentle rotation and shaking.</li>
	<li>Repeat step 3.5 through step 3.6 one more time after another 20 min. Put the supernatant into the respective 50 mL centrifuge tube from step 3.5.</li>
	<li>Stop the digestion after 60 min by quenching the digestion buffer with ice-cold FACS buffer.</li>
	<li>Filter the digestion suspension and supernatant through a 40 &#181;m cell strainer on top of 50 mL centrifuge tubes. Top up to 40 mL with FACS buffer.</li>
	<li>Centrifuge the cell suspension at 500 x g for 8 min at 4 &#176;C. Decant the supernatant and resuspend the cell pellet in 3 mL of ACK lysis buffer. Incubate on ice for 5 min and top up to 40 mL with FACS buffer.</li>
	<li>Centrifuge at 500 x g for 5 min at 4 &#176;C. Decant the supernatant.</li>
	<li>Resuspend in 0.5 mL of FACS buffer, pipetting up and down to ensure cells are evenly dispersed.</li>
	<li>Filter the suspension through a 40 &#181;m filter on 5 mL polystyrene round-bottom tubes with a cell-strainer cap.</li>
</ol>
4. Staining for Fluorescence-activated cell sorting (FACS)
<ol>
	<li>For single color and fluorescence-minus-one (FMO) controls, prepare 30 &#181;L of staining buffer (antibodies diluted in FACS buffer) at 2x working concentration in pre-labeled 1.5 mL centrifuge tubes. Refer to Table 1 for antibody staining buffer setup.</li>
	<li>Prepare single colors and FMO controls for muscle and heart separately.</li>
	<li>Transfer 30 &#181;L of single color and FMO control staining buffer to a 96-well plate. Top up with 20 &#181;L of FACS buffer.</li>
	<li>For sample FACS staining, prepare 0.5 mL of staining buffer (antibody diluted in FACS buffer) at 2x working concentration in 1.5 mL centrifuge tubes.</li>
	<li>Add 10 &#181;L of the cell suspension to single color and FMO control wells. Add staining buffer to the rest of the cell suspension and resuspend.</li>
	<li>Incubate at 4 &#176;C, protected from light for 30 min.</li>
	<li>Top up the wells with 100 &#181;L of FACS buffer, and the sample tube with 2 mL of FACS buffer.</li>
	<li>Centrifuge at 500 x g for 5 min at 4 &#176;C and remove the supernatant.</li>
	<li>Repeat steps 4.7 and 4.8.</li>
	<li>Resuspend the cell pellet in the viability buffer. 100 &#181;L for single color and FMO controls, 1 mL for FACS sorting samples.</li>
	<li>Prepare the collection tubes by adding 300 &#181;L of proliferation media in 1.7 mL centrifuge tubes.</li>
</ol>
5. Fluorescence-activated cell sorting
<ol>
	<li>Gating strategy: Use FSC vs. SSC (log) to remove debris. Use FSC-H vs. FSC-A to gate singlets. Use CD31 vs. PI to gate on viable and lineage-negative cells. Use PDGFR&#945; to gate for FAPs (Figure 2).</li>
	<li>Sort into collection tubes previously prepared.</li>
</ol>
6. Tissue culture
<ol>
	<li>Centrifuge the collected cells at 800 x g for 10 min at 4 &#176;C.</li>
	<li>Remove the supernatant with a P1000 pipette. Be careful not to disturb the cell pellet.</li>
	<li>Resuspend the pellet in culture media (250 &#181;L/well) and seed at 25k cells/well in a 48-well tissue culture plate.</li>
	<li>Culture in an incubator (37 &#176;C, 5% CO2). Change media every 3 days until cells reach 80% confluency.</li>
</ol>
7. Fibrogenic differentiation
<ol>
	<li>Once the cells are ready to be treated, place basic media and 1x DPBS in a warm bath or at room temperature before using them.</li>
	<li>Aspirate the culture media.</li>
	<li>Rinse with 200 &#181;L of 1x DPBS twice.</li>
	<li>Rinse with 200 &#181;L of basic media once.</li>
	<li>Add 250 &#181;L of fibrogenic or basic media to the respective wells.</li>
	<li>Return the cells to the incubator (37 &#176;C, 5% CO2).</li>
	<li>Stop the differentiation after 48 h.</li>
</ol>
8. Adipogenic differentiation
<ol>
	<li>Once the cells are ready to be treated, place basic media, adipogenic media, and 1x DPBS in a warm bath or at room temperature before using them.</li>
	<li>Aspirate the culture media.</li>
	<li>Rinse with 200 &#181;L of 1x DPBS twice.</li>
	<li>Rinse with 200 &#181;L of basic media once.</li>
	<li>Add 400 &#181;L of adipogenic or basic media to respective wells.</li>
	<li>Return to incubator (37&#176;C, 5% CO2).</li>
	<li>Every 2 days, remove 200 &#181;L media with a pipette from the wells and add 200 &#181;L of fresh adipogenic or basic media.</li>
	<li>Stop differentiation on the 6th day for skeletal muscle FAPs, and 14th day for cardiac FAPs.</li>
</ol>
9. Immunostaining
<ol>
	<li>Stop the differentiation assay by aspirating media and rinsing with 250 &#181;L of 1x PBS twice. Perform all washes with 18 G needle attached to 3 mL syringe to avoid cell detachment.</li>
	<li>Add 250 &#181;L of 4% PFA per well and incubate at room temperature for 7 min.</li>
	<li>Remove the PFA and rinse 3 times for 5 min each time with 250 &#181;L of 1x PBS.</li>
	<li>On the last wash, aspirate 1x PBS and add 250 &#181;L of Donkey + MOM blocking buffer.</li>
	<li>Incubate at room temperature for 60 min.</li>
	<li>Prepare primary antibody staining solution in Donkey blocking buffer (anti-SMA and anti-perilipin) for a final volume of 100 &#181;L per well.</li>
	<li>Aspirate the staining wells. Leave Donkey + MOM blocking buffer in control well if applicable.</li>
	<li>Add primary antibody staining solution in the respective wells.</li>
	<li>Incubate at 4 &#176;C overnight.</li>
	<li>Next day, wash 3 times for 5 min each time with 1x PBS.</li>
	<li>Prepare secondary antibody staining solution in Donkey blocking buffer with a final volume of 100 &#181;L per well.</li>
	<li>Aspirate the last wash and add secondary antibody staining solution in all wells.</li>
	<li>Incubate at room temperature for 45 min (samples should be protected from light from this point forward).</li>
	<li>Wash 3 times for 5 min each time with 1x PBS.</li>
	<li>Aspirate the last wash and add 250 &#181;L of DAPI staining solution.</li>
	<li>Incubate at room temperature for 5 min.</li>
	<li>Remove the DAPI staining solution and wash once for 5 min with 1x PBS.</li>
	<li>Add 100 &#181;L of Fluoromount-G in each well.</li>
	<li>Seal the plate with the lid and paraffin film to minimize evaporation. Store at 4 &#176;C, protected from the light until imaging.</li>
	<li>Image with a microscope (Figure 3 and Figure 4). 
	NOTE: Donkey serum is used in blocking buffer as a species-specific block against the Donkey raised secondary antibodies that are used in this protocol. The mouse-on-mouse blocking reagent is used against the anti-SMA primary antibody that is raised in the mouse.</li>
</ol>

Mouse Heart and Skeletal Muscle Digestion for Fibro-Adipogenic Progenitor Culture

<ol>
	<li>Gaire, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spiny+mouse+(Acomys):+An+emerging+research+organism+for+regenerative+medicine+with+applications+beyond+the+skin.">Spiny mouse (Acomys): An emerging research organism for regenerative medicine with applications beyond the skin.</a> NPJ Regen Med. 6, 1 (2021).</li><li>Sandoval, A. G. W., Maden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+in+the+spiny+mouse,+Acomys,+a+new+mammalian+model.">Regeneration in the spiny mouse, Acomys, a new mammalian model.</a> Curr. Opin. Genet. Dev. 64, 31-36 (2020).</li><li>Judson, R. N., Zhang, R. H., Rossi, F. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-resident+mesenchymal+stem/progenitor+cells+in+skeletal+muscle:+Collaborators+or+saboteurs.">Tissue-resident mesenchymal stem/progenitor cells in skeletal muscle: Collaborators or saboteurs.</a> FEBS J. 280 (17), 4100-4108 (2013).</li><li>Uezumi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrosis+and+adipogenesis+originate+from+a+common+mesenchymal+progenitor+in+skeletal+muscle.">Fibrosis and adipogenesis originate from a common mesenchymal progenitor in skeletal muscle.</a> J Cell Sci. 124, 3654-3664 (2011).</li><li>Joe, A. W. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+injury+activates+resident+fibro/adipogenic+progenitors+that+facilitate+myogenesis.">Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis.</a> Nat Cell Biol. 12, 153-163 (2010).</li><li>Uezumi, A., Fukada, S. I., Yamamoto, N., Takeda, S., Tsuchida, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+progenitors+distinct+from+satellite+cells+contribute+to+ectopic+fat+cell+formation+in+skeletal+muscle.">Mesenchymal progenitors distinct from satellite cells contribute to ectopic fat cell formation in skeletal muscle.</a> Nat Cell Biol. 122 (12), 143-152 (2010).</li><li>Soliman, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenic+Potential+of+Hic1-Expressing+Cardiac+Stromal+Progenitors.">Pathogenic Potential of Hic1-Expressing Cardiac Stromal Progenitors.</a> Cell Stem Cell. 26 (2), 205-220 (2020).</li><li>Hall, C., Gehmlich, K., Denning, C., Pavlovic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+relationship+between+cardiac+fibroblasts+and+cardiomyocytes+in+health+and+disease.">Complex relationship between cardiac fibroblasts and cardiomyocytes in health and disease.</a> J Am Heart Assoc. 10, 1-15 (2021).</li><li>Gawriluk, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+ear-hole+closure+identifies+epimorphic+regeneration+as+a+discrete+trait+in+mammals.">Comparative analysis of ear-hole closure identifies epimorphic regeneration as a discrete trait in mammals.</a> Nat Commun. 71 (7), 1-16 (2016).</li><li>Seifert, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+shedding+and+tissue+regeneration+in+African+spiny+mice+(Acomys).">Skin shedding and tissue regeneration in African spiny mice (Acomys).</a> Nature. 489, 561 (2012).</li><li>Koopmans, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemic+tolerance+and+cardiac+repair+in+the+spiny+mouse+(Acomys).">Ischemic tolerance and cardiac repair in the spiny mouse (Acomys).</a> NPJ Regen Med. 6, 78 (2021).</li><li>Peng, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+spiny+mice+(Acomys)+exhibit+endogenous+cardiac+recovery+in+response+to+myocardial+infarction.">Adult spiny mice (Acomys) exhibit endogenous cardiac recovery in response to myocardial infarction.</a> NPJ Regen Med. 6, 74 (2021).</li><li>Maden, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfect+chronic+skeletal+muscle+regeneration+in+adult+spiny+mice,+Acomys+cahirinus.">Perfect chronic skeletal muscle regeneration in adult spiny mice, Acomys cahirinus.</a> Sci Rep. 8, 8920 (2018).</li><li>Riparini, G., Simone, J. M., Sartorelli, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FACS-isolation+and+culture+of+fibro-adipogenic+progenitors+and+muscle+stem+cells+from+unperturbed+and+injured+mouse+skeletal+muscle.">FACS-isolation and culture of fibro-adipogenic progenitors and muscle stem cells from unperturbed and injured mouse skeletal muscle.</a> J Vis Exp. (184), e63983 (2022).</li><li>Low, M., Eisner, C., Rossi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibro/adipogenic+progenitors+(FAPs):+Isolation+by+FACS+and+culture.">Fibro/adipogenic progenitors (FAPs): Isolation by FACS and culture.</a> Methods Mol Biol. 1556, 179-189 (2017).</li><li>Molina, T., Fabre, P., Dumont, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibro-adipogenic+progenitors+in+skeletal+muscle+homeostasis,+regeneration+and+diseases.">Fibro-adipogenic progenitors in skeletal muscle homeostasis, regeneration and diseases.</a> Open Biol. 11 (12), 210110 (2021).</li><li>Contreras, O., Rossi, F. M. V., Theret, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins,+potency,+and+heterogeneity+of+skeletal+muscle+fibro-adipogenic+progenitors-time+for+new+definitions.">Origins, potency, and heterogeneity of skeletal muscle fibro-adipogenic progenitors-time for new definitions.</a> Skelet Muscle. 111 (11), 1-25 (2021).</li><li>Fitzgerald, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MME++fibro-adipogenic+progenitors+are+the+dominant+adipogenic+population+during+fatty+infiltration+in+human+skeletal+muscle.">MME+ fibro-adipogenic progenitors are the dominant adipogenic population during fatty infiltration in human skeletal muscle.</a> Commun Biol. 61 (6), 1-21 (2023).</li><li>Babaeijandaghi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DPPIV++fibro-adipogenic+progenitors+form+the+niche+of+adult+skeletal+muscle+self-renewing+resident+macrophages.">DPPIV+ fibro-adipogenic progenitors form the niche of adult skeletal muscle self-renewing resident macrophages.</a> Nat Commun. 141 (14), 1-10 (2023).</li><li>Schutz, P. W., Cheung, S., Yi, L., Rossi, F. M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+activation+patterns+of+CD10++fibro-adipogenic+progenitors+across+acquired+disease+states+in+human+skeletal+muscle+biopsies.">Cellular activation patterns of CD10+ fibro-adipogenic progenitors across acquired disease states in human skeletal muscle biopsies.</a> Free Neuropathol. 5, 3-3 (2024).</li></ol>

The authors have no conflict of interest to disclose.

Spiny mouse tissues are more sensitive to the stresses in tissue dissociation, and there are several aspects of this protocol aimed at minimizing stress to improve cell viability. Serial enzymatic dissociation technique is employed for sample preparation to lower the concentration of the enzyme required. As enzymatic digestion proceeds, the enzymatic activity decreases. By replacing it with fresh enzymes, consistent enzymatic activity throughout the entire digestion can be better achieved, and high concentrations of the ...

Initially recognized for its exceptionally fragile skin and remarkable ability to repair skin injuries, spiny mouse has demonstrated superior regenerative capacity in various organ systems, such as musculoskeletal, renal, central nervous system, and cardiovascular, when compared to Mus musculus1,2.
Fibro-adipogenic progenitors (FAPs) are a subset of stromal cells that are resident in various tissues, including skeletal and cardiac muscle. These cells possess a unique capacity to commit to fibrogenic and adipogenic lineages in vivo and in vitro3,4. FAPs play a crucial role in tissue regeneration by modulating the extracellular matrix and supporting the functions of other cell types involved in the repair process. In skeletal muscle, FAPs become activated in response to injury and facilitate muscle stem cell differentiation and myogenesis5,6. In ischemic injury to the heart, FAPs lay down scar tissue to maintain the integrity of the myocardium. In contrast to muscle, cardiac FAPs become chronically activated and contribute to pathological remodeling7,8.
Previous studies have demonstrated that spiny mouse extracellular matrix has different composition, structure, and properties that support regeneration in comparison to Mus musculus9,10. In muscle and heart injuries, FAPs have been noted to contribute to superior healing in spiny mouse11,12,13. Understanding the behavior and regulatory control of spiny mouse FAPs and stroma may shed light on the mechanism behind their regenerative capabilities. While FAPs are extensively studied in other animal models, such as in mus musculus14,15, currently, there are no published protocols for isolating spiny mouse FAPs. Developing such a protocol would fill a significant gap in the field and enable researchers to examine the cellular and molecular mechanisms underlying the spiny mouse&#39;s regenerative potential.
This protocol describes a robust and reproducible protocol for isolating, expanding, and differentiating skeletal and cardiac muscle FAPs from spiny mouse. The protocol described here yields high-quality single-cell suspensions that are suitable for fluorescence-activated cell sorting (FACS). By using rh-TGF&#946;1 or a compatible commercially available media, two methods widely used to differentiate mus musculus FAPs16, the sorted spiny FAPs maintain their capacity to differentiate along the fibrogenic lineage and adipogenic lineage, respectively (Figure 1).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5M EDTA</td>
			<td>Invitrogen</td>
			<td>15575&#8211;038</td>
			<td></td>
		</tr>
		<tr>
			<td>1.7 mL Microcentrifuge tubes</td>
			<td>VWR</td>
			<td>87003-294</td>
			<td></td>
		</tr>
		<tr>
			<td>15&#160;mL centrifuge tube</td>
			<td>Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>1x Dulbecco&#8217;s Phosphate Buffered Saline (DPBS)</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL syringe</td>
			<td>BD</td>
			<td>309661</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8242;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Invitrogen</td>
			<td>D3571</td>
			<td></td>
		</tr>
		<tr>
			<td>40&#160;&#956;m cell strainer</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>48 well flat-bottom tissue culture plate</td>
			<td>Falcon</td>
			<td>53078</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL polypropylene</td>
			<td>Falcon</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL polystyrene round-bottom tube with cell-strainer cap</td>
			<td>Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL syringe</td>
			<td>BD</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>50&#160;mL centrifuge tube</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Petri dish</td>
			<td>Falcon</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well V-bottom tissue culture plate</td>
			<td>Corning</td>
			<td>3894</td>
			<td></td>
		</tr>
		<tr>
			<td>Acomys dimidiatus mice (spiny mice)</td>
			<td></td>
			<td></td>
			<td>kindly gifted by Dr. Ashley W. Seifert (University of Kentucky).</td>
		</tr>
		<tr>
			<td>Ammonium-chloride-potassium (ACK) lysing buffer</td>
			<td>Gibco</td>
			<td>A10492-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-perilipin (1:100)</td>
			<td>Sigma</td>
			<td>P1873</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-SMA (1:100)</td>
			<td>Invitrogen</td>
			<td>14-9760-82</td>
			<td></td>
		</tr>
		<tr>
			<td>APC PDGFRa (1:800)</td>
			<td>Abcam</td>
			<td>ab270085</td>
			<td></td>
		</tr>
		<tr>
			<td>BD PrecisionGlide Needle 18 G</td>
			<td>BD</td>
			<td>305195</td>
			<td></td>
		</tr>
		<tr>
			<td>BD PrecisionGlide Needle 23 G</td>
			<td>BD</td>
			<td>305145</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A7906-100g</td>
			<td></td>
		</tr>
		<tr>
			<td>BV605 CD31 (1:500)</td>
			<td>BD biosciences</td>
			<td>744359</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C4901</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-mouse Alexa 555 (1:1000)</td>
			<td>Invitrogen</td>
			<td>A31570</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit Alexa 647 (1:1000)</td>
			<td>Invitrogen</td>
			<td>A31573</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Sigma</td>
			<td>S30-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS sorter - MoFlo Astrios 5 lasers</td>
			<td>Beckman coulter</td>
			<td>B52102</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gemini</td>
			<td>100-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine scissors</td>
			<td>FST</td>
			<td>14058-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>SouthernBiotech</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>FST</td>
			<td>11051-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostat</td>
			<td>FST</td>
			<td>91308-12</td>
			<td></td>
		</tr>
		<tr>
			<td>human FGF-basic recombinant protein (bFGF)</td>
			<td>Gibco</td>
			<td>13256029</td>
			<td></td>
		</tr>
		<tr>
			<td>Human TGF beta 1 recombinant protein (TGFb1)</td>
			<td>eBiosciences</td>
			<td>14-8348-62</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator - Heracell 160i CO2</td>
			<td>ThermoFisher</td>
			<td>51033557</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope - Revolve</td>
			<td>ECHO</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Liberase</td>
			<td>Roche</td>
			<td>5401127001</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse MesenCult Adipogenic Differentiation 10x Supplement</td>
			<td>STEMCELL technologies</td>
			<td>5507</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse on mouse (MOM) blocking reagent</td>
			<td>Vector Laboratories</td>
			<td>MKB-2213</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P1648-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140&#8211;122</td>
			<td></td>
		</tr>
		<tr>
			<td>PicoLab Mouse Diet 20</td>
			<td>LabDiet</td>
			<td>3005750-220</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide (PI)</td>
			<td>Invitrogen</td>
			<td>P3566</td>
			<td></td>
		</tr>
		<tr>
			<td>Transport vial 5mL tube</td>
			<td>Caplugs Evergreen</td>
			<td>222-3005-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>9036-19-5</td>
			<td></td>
		</tr>
	</tbody>
</table>

fibro-adipogenic progenitor isolation, expansion, and differentiation from the spiny mouse model

Due to its exceptional repair program, the spiny mouse is an emerging research model for regenerative medicine. Fibro-adipogenic progenitors are tissue-resident cells that are able to differentiate into adipocytes, fibroblasts, and chondrocytes. Fibro-adipogenic progenitors are fundamental for orchestrating tissue regeneration as they are responsible for extracellular matrix remodeling after injury. This study focuses on investigating the specific role of fibro-adipogenic progenitors in spiny mouse cardiac repair and skeletal muscle regeneration. To this end, a protocol has been optimized for the purification of spiny mouse fibro-adipogenic progenitors by flow cytometry from enzymatically dissociated skeletal and cardiac muscle. The population obtained from this protocol is capable of expanding in vitro, and can be differentiated to myofibroblasts and adipocytes. This protocol offers a valuable tool for researchers to examine the distinctive properties of spiny mouse, and to compare them to the Mus musculus. This will provide insights that could advance the understanding of regenerative mechanisms in this intriguing model.

Author Spotlight: Streamlining Cell Analysis with an Innovative Protocol for Studying Spiny Mouse Models

Fibro-Adipogenic Progenitor Isolation, Expansion, and Differentiation from the Spiny Mouse Model

Our overarching vision is to better understand and grasp cell communication and cell behavior leading to tissue metastasis and how the cellular processes are misregulated in fed repair. To do so, we are using various models of which one is the Spiny mouse. One of the main issues with this new mouse model is the lack of tools to study it.Here we provide a new protocol, which contains reliable enzyme digestion and antibody staining. Allowing us to successfully sort fibro-adipogenic progenitors and culture them in vitro for further analysis of their fate. We have tested various enzyme combinations and concentrations, as well as multiple antibodies, and our method provides references for the most reliable and robust protocol.As we now have a reliable enzymatic digestion, we will continue to test for other markers and antibodies to successfully analyze endothelial cells, pericytes, immune cells, and myogenic cells.

The schematic for this protocol to isolate and culture skeletal muscle and cardiac FAPs is summarized in Figure 1. For tissue collection, the liver changing color from dark red to pale yellow is usually indicative of a successful perfusion. With the specified age ranges of spiny mouse, the heart weights are typically around 200 mg, while the quadricep muscle is around 350 mg.
During each digestion buffer change in steps 3.5-3.7, the digestion buffer in the digesti...

This protocol outlines the isolation of skeletal and cardiac muscle fibro-adipogenic progenitors (FAPs) from spiny mouse (Acomys) via enzymatic dissociation and fluorescence-activated cell sorting. The FAPs obtained from this protocol can be effectively expanded and differentiated to myofibroblasts and adipocytes.

Due to its exceptional repair program, the spiny mouse is an emerging research model for regenerative medicine. Fibro-adipogenic progenitors are ...

fibro-adipogenic-progenitor-isolation-expansion-differentiation-from

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Research

JoVE Journal

Developmental Biology

Collection of Skeletal and Cardiac Muscles from Spiny Mouse for Isolating Fibro-Adipogenic Progenitors (FAPs)

To begin place a euthanized mouse on a dissection stage in a supine position, thoroughly spray the mouse's fur with 70%ethanol. Make a two to three centimeter long vertical incision at the ventral midline. Expose the ribcage and the abdominal muscle.Then use tissue scissors to cut through the muscle at the xiphoid process and expose the diaphragm. Now cut the diaphragm and rib cage on both sides. Clamp and peel back the cut ribs with a hemostat.Nick the right atrium with tissue scissors. Then insert a 23 gauge needle attached to a 20 milliliter syringe into the apex of the heart. Slowly inject 20 milliliters of two millimolar PBSEDTA into the heart.Excise it and place it in a 60 millimeter Petri dish containing cold PBS on ice. Remove the atria and rinse out as much blood as possible. Next, remove the skin above the knee joint of the mouse.With a pair of fine forceps, remove the fascia. Use scissors to isolate the quadriceps with the femur as a guide. Then transfer the quadriceps muscle to a separate Petri dish containing cold PBS.

Digestion of Mouse Skeletal and Cardiac Muscles for Fluorescence-Activated Cell Sorting and Culture of Fibro-Adipogenic Progenitors (FAPs)

To begin, add two milliliters of digestion buffer to a five milliliter transport vial containing the mouse heart tissue, and four milliliters of digestion buffer into a 15 milliliter centrifuge tube containing the mouse skeletal tissue. Place the heart and quadricep muscle tissue isolated from a spiny mouse onto the lid of a Petri dish. With a pair of tissue scissors, mince it into pieces smaller than one cubic millimeter.Transfer the minced tissue into the respective digestion tubes. Vortex the contents of the tubes for two seconds at low speed. After 20 minutes, remove the tubes off the rotator.Allow the tissue pieces to settle. Use a P 1000 pipette to aspirate the supernatant. Transfer the supernatant into 50 milliliter centrifuge tubes containing 20 milliliters of ice cold FACS buffer.Top up the digestion tubes with the respective volumes of digestion buffer. Incubate them on the rotator again. After subjecting the muscles to another round of digestion, add ice cold FACS buffer to quench the digestion.Place a 40 micrometer cell strainer on top of a 50 milliliter centrifuge tube. Filter the digestion suspension and supinate through the strainer. Centrifuge the cell suspension at 500 G for eight minutes at four degrees Celsius.After removing the supernatant, add three milliliters of ACK lysis buffer to the pellet and resuspend. Incubate the suspension for five minutes on ice. Top up the volume to 40 milliliters with FACS buffer.Centrifuge the samples again at 500 G for five minutes at four degrees celsius. After decanting the supernatant resuspend the pellet in 0.5 milliliters of FACS buffer. Finally, filter the suspension through a 40 micrometer cell strainer cap, placed on a five milliliter polystyrene round bottom tube.

Fluorescence-Activated Cell Sorting and Culture of Fibro-Adipogenic Progenitors (FAPs)

To begin, prepare 30 microliters of staining buffer at 2x working concentrations for single color and FMO controls. Transfer the single color and FMO control staining buffer to the wells of a 96 well plate. Top up the volume with 20 microliters of FACS buffer.To stain the sample, prepare 0.5 milliliters of 2x FACS staining buffer in 1.5 milliliter centrifuge tubes. Pipette 10 microliters of the cell suspension to single color and FMO control wells. Then, add 2x FACS staining buffer to the rest of the cell suspension and incubate.Next, add 100 microliters of FACS buffer into the wells and two milliliters of the buffer into the sample tubes. Centrifuge at 500G for five minutes at four degrees Celsius. Aspirate the supernatant, then resuspend the cell pellet in viability buffer.Add 300 microliters of proliferation media into 1.5 milliliter centrifuge tubes. These will act as collection tubes for cell sorting. After sorting the cells using FACS, centrifuge the collected cells at 800G for 10 minutes at four degrees Celsius.Use a P1000 pipette to carefully remove the supernatant without disturbing the pellet. Resuspend the pellet in proliferation media to a final density of 100 cells per microliter. Seed the cell suspension into the wells of a 48 well tissue culture plate.Finally transfer the plate to an incubator at 37 degrees Celsius under respective gas supplementation until cells are confluent. Confluent cells with typical fibroblast morphology were obtained within five days of culture.

1.1K Views.  University of British Columbia.  This protocol outlines the isolation of skeletal and cardiac muscle fibro-adipogenic progenitors (FAPs) from spiny mouse (Acomys) via enzymatic dissociation and fluorescence-activated cell sorting. The FAPs obtained from this protocol can be effectively expanded and differentiated to myofibroblasts and adipocytes. 

Mouse Heart and Skeletal Muscle Digestion for Fibro-Adipogenic Progenitor Culture

Summary