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...

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>

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.