Name: Video: Auteur in de schijnwerpers: celanalyse stroomlijnen met een innovatief protocol voor het bestuderen van stekelmuismodellen
Uploaded: 2024-11-15T12:20:00.000+00:00
Duration: 7 min

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

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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. 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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 enzyme are avoided. Furthermore, changing the digestion buffer to over-digest the already released cells in the solution is avoided. Secondly, ACK lysis buffer is used to remove red blood cells. Although the heart is exsanguinated and perfused, it still contains red blood cells as it is a highly vascularized organ. By removing contaminating red blood cells, the time required for FACS is minimized to avoid cell degradation. Lastly, it is important to work fast and diligently from step 2.1 to step 6.4, and follow best practices for working with cells, such as gentle pipetting and keeping on ice as much as possible. Some details are difficult to show even in the audiovisual format and may require a certain level of experience.
In cell culture, the initial seeding density is critical for optimal cell expansion. FAPs in the dish communicate in a paracrine fashion and stimulate growth via secreted growth factors. In low seeding density conditions, the cells do not receive sufficient signals for growth and will either fail to expand or require additional culturing time to reach appropriate confluency levels for downstream applications. This is also the rationale for changing only half of the media during each media change in adipogenic differentiation. It is important to leave some conditioned media that contains the growth factors from pre-adipocytes that are critical for their differentiation.
During the immunostaining, it is important to be gentle when adding or removing buffers to minimize cell detachment. In particular, in step 9.1, the cells are the most prone to detach prior to fixation. Manual aspiration, such as with a pipette or a needle and syringe, is recommended, but the use of powerful vacuum aspirators is not advised.
A limitation of this protocol is that PDGFR&#945; is a general marker of the FAPs, and more markers are required to distinguish the unique subtypes of FAPs17. Subtypes such as DPP4+ progenitors like FAPs and CD10+ adipogenic FAPs have been implicated in both disease and homeostatic context18,19,20. FAP subsets likely also exist in spiny mouse. They may exist in different proportions, exhibit different cellular behavior, or have different physiological functions compared to corresponding mus musculus FAP subsets. Further optimization of FAP subtype-specific markers in spiny mouse will be needed to examine the details of spiny mouse FAPs.
In summary, this protocol details a robust and reproducible method of isolating, culturing, and differentiating skeletal muscle and cardiac FAPs from spiny mouse. The spiny mouse is gathering momentum as a new hyper-regenerative animal model. FAPs and the stroma are highly implicated in maintaining muscle and cardiac homeostasis and contribute to organ dysfunction in multiple chronic disease models5,7. Characterizing spiny mouse FAPs may elucidate the mechanism behind their remarkable regenerative capacity and pave the way toward novel strategies in regenerative medicine.

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><tbody><tr><td>0.5M EDTA</td><td>Invitrogen</td><td>15575&amp;ndash;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&amp;nbsp;mL centrifuge tube</td><td>Falcon</td><td>352096</td><td></td></tr><tr><td>1x Dulbecco&amp;rsquo;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&amp;prime;,6-diamidino-2-phenylindole (DAPI)</td><td>Invitrogen</td><td>D3571</td><td></td></tr><tr><td>40&amp;nbsp;&amp;mu;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&amp;nbsp;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>&lt;em&gt;Acomys dimidiatus&lt;/em&gt; 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>CaCl&lt;sub&gt;2&lt;/sub&gt;</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 CO&lt;sub&gt;2&lt;/sub&gt;</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&amp;ndash;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.

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 digestion tubes appears opaque as cells are released from the tissue. At the end of the digestion, the solution must be a relatively homogenous slurry with some tissue pieces remaining. Be sure to end the digest before all tissues are completely digested to avoid over-digestion, which will negatively impact the viability of the cells and culture outcomes.
The expected flow plots for FACS are shown in Figure 2. FAPs are expected to represent around 2% of live and LIN- cells. The percentage of PI+ events is an indicator of the quality of the sample preparation and should be less than 10%. In this study, 150k FAPs per heart (ventricles only) and 100k FAPs from two quadricep muscles are typically obtained.
Once in the plate, cells typically attach within 72 h, reach 80% confluency within 5 days, and display typical fibroblast morphology with projections (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66717/66717fig01.jpg" /> 
Figure 1: Schematic representation of the protocol and approximate time required for each step. Tissue collection: 10 min per mouse. Tissue digestion: 1.5-2 h. Cell staining: 45 min-75 min. Fluorescence-activated cell sorting: 2 h. Fibro-adipogenic progenitors (FAP) expansion: 4 days. FAP differentiation: 48 h for fibrogenic differentiation, 6 days for adipogenic differentiation for skeletal muscle FAPs, and 14 days for cardiac FAPs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66717/66717fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66717/66717fig02.jpg" /> 
Figure 2: Representative FACS plots for skeletal muscle (top) and cardiac (bottom) FAP isolation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66717/66717fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66717/66717fig03.jpg" /> 
Figure 3: Spiny mouse skeletal muscle FAP differentiation. (A) Fibrogenic differentiation control. (B) Fibrogenic differentiation. (C) Adipogenic differentiation control. (D) Adipogenic differentiation. Perilipin (green), SMA (magenta), DAPI (blue), scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66717/66717fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66717/66717fig04.jpg" /> 
Figure 4: Spiny mouse cardiac FAP differentiation. (A) Fibrogenic differentiation control. (B) Fibrogenic differentiation. (C) Adipogenic differentiation control. (D) Adipogenic differentiation. Perilipin (green), SMA (magenta), DAPI (blue), scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66717/66717fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66717/66717fig05.jpg" /> 
Figure 5: Brightfield images of spiny mouse skeletal muscle (A) and heart (B) FAPs after 5 days of culture, before differentiation. Scale bar = 360 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66717/66717fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Controls</td>
			<td>CD31 SC</td>
			<td>PDGFR&#945; SC</td>
			<td>CD31 FMO</td>
			<td>PDGFR&#945; FMO</td>
			<td>Sample</td>
		</tr>
		<tr>
			<td>CD31 antibody</td>
			<td>+</td>
			<td>-</td>
			<td>-</td>
			<td>+</td>
			<td>+</td>
		</tr>
		<tr>
			<td>PDGFR&#945; antibody</td>
			<td>-</td>
			<td>+</td>
			<td>+</td>
			<td>-</td>
			<td>+</td>
		</tr>
	</tbody>
</table>
Table 1: Schematic guide for setting up antibody staining cocktails for controls and sample. SC = single color control, FMO = fluorescence minus one control, + = with the corresponding antibody, - = without the corresponding antibody.

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

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.

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 ...

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

Nanyang Technological University

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Research

JoVE Journal

Developmental Biology

4.1K Views.  University of British Columbia. Onze overkoepelende visie is om celcommunicatie en celgedrag dat leidt tot weefselmetastase beter te begrijpen en te begrijpen en hoe de cellulaire processen verkeerd worden gereguleerd bij gevoed herstel. Hiervoor gebruiken we verschillende modellen, waaronder de Spiny mouse. Een van de belangrijkste problemen met dit nieuwe muismodel is het gebrek aan hulpmiddelen om het te bestuderen.Hier bieden we een nieuw protocol, dat betrouwbare enzymvertering ...

Video: Auteur in de schijnwerpers: celanalyse stroomlijnen met een innovatief protocol voor het bestuderen van stekelmuismodellen

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting

Expansion of Perivascular Adipose Tissue (PVAT), a major regulator of vascular function through paracrine signaling, is directly related to the development of hypertension during obesity. The extent of hypertrophy and hyperplasia depends on depot location, sex, and the type of Adipocyte Progenitor Cell (APC) phenotypes present. Techniques used for APC and preadipocytes isolation in the last 10 years have drastically improved the accuracy at which individual cells can be identified based on specific cell surface markers. However, isolation of APC and adipocytes can be a challenge due to the fragility of the cell, especially if the intact cell must be retained for cell culture applications.
Magnetic-activated Cell Sorting (MCS) provides a method of isolating greater number of viable APC per weight unit of adipose tissue. APC harvested by MCS can be used for in vitro protocols to expand preadipocytes and differentiate them into adipocytes through use of growth factor cocktails allowing for analysis of the prolific and adipogenic potential retained by the cells. This experiment focused on the aortic and mesenteric PVAT depots, which play key roles in the development of cardiovascular disease during expansion. These protocols describe methods to isolate, expand, and differentiate a defined population of APC. This MCS protocol allows isolation to be used in any experiment where cell sorting is needed with minimal equipment or training. These techniques can aid further experiments to determine the functionality of specific cell populations based on the presence of cell surface markers.

Here we report a method for isolation of Adipocyte Progenitor Cell (APC) populations from Perivascular Adipose Tissue (PVAT) using Magnetic-activated Cell Sorting (MCS). This method allows for an increased isolation of APC per gram of adipose tissue when compared to Fluorescence-Activated Cell Sorting (FACS).

Expansion of Perivascular Adipose Tissue (PVAT), a major regulator of vascular function through paracrine signaling, is directly related to the ...

FACS-Isolation and Culture of Fibro-Adipogenic Progenitors and Muscle Stem Cells from Unperturbed and Injured Mouse Skeletal Muscle

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along fibrogenic, adipogenic, osteogenic, or chondrogenic lineage. Together with muscle stem cells (MuSCs), FAPs play a critical role in muscle homeostasis, repair, and regeneration, while actively maintaining and remodeling the extracellular matrix (ECM). In pathological conditions, such as chronic damage and muscular dystrophies, FAPs undergo aberrant activation and differentiate into collagen-producing fibroblasts and adipocytes, leading to fibrosis and intramuscular fatty infiltration. Thus, FAPs play a dual role in muscle regeneration, either by sustaining MuSC turnover and promoting tissue repair or contributing to fibrotic scar formation and ectopic fat infiltrates, which compromise the integrity and function of the skeletal muscle tissue. A proper purification of FAPs and MuSCs is a prerequisite for understanding the biological role of these cells in physiological as well as in pathological conditions. Here, we describe a standardized method for the simultaneous isolation of FAPs and MuSCs from limb muscles of adult mice using fluorescence-activated cell sorting (FACS). The protocol describes in detail the mechanical and enzymatic dissociation of mononucleated cells from whole limb muscles and injured tibialis anterior (TA) muscles. FAPs and MuSCs are subsequently isolated using a semi-automated cell sorter to obtain pure cell populations. We additionally describe an optimized method for culturing quiescent and activated FAPs and MuSCs, either alone or in coculture conditions.

The precise identification of fibro-adipogenic progenitor cells (FAPs) and muscle stem cells (MuSCs) is critical to studying their biological function in physiological and pathological conditions. This protocol provides guidelines for the isolation, purification, and culture of FAPs and MuSCs from adult mouse muscles.

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along ...

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

Immunology and Infection

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident adipose progenitor cells (APCs) play a key role in adipose tissue homeostasis and can contribute to the tissue pathology. The growing use of single cell analysis technologies &#8211; including single-cell RNA-sequencing and single-cell proteomics &#8211; is transforming the stem/progenitor cell field by permitting unprecedented resolution of individual cell expression changes within the context of population- or tissue-wide changes. In this article, we provide detailed protocols to dissect mouse epididymal adipose tissue, isolate single adipose tissue-derived cells, and perform fluorescence activated cell sorting (FACS) to enrich for viable Sca1+/CD31-/CD45-/Ter119- APCs. These protocols will allow investigators to prepare high quality APCs suitable for downstream analyses such as single cell RNA sequencing.

We present a simple method to isolate highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting.

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...

Biology

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white preadipocyte cell lines. These cultured cell lines, however, have limitations. They do not fully capture the diverse functional spectrum of the heterogenous adipocyte populations that are now known to exist within white adipose depots. To provide a more physiologically relevant model to study the complexity of white adipose tissue, a protocol has been developed and optimized to enable simultaneous isolation of primary white and brown adipocyte progenitors from newborn mice, their rapid expansion in culture, and their differentiation in vitro into mature, fully functional adipocytes. The primary advantage of isolating primary cells from newborn, rather than adult mice, is that the adipose depots are actively developing and are, therefore, a rich source of proliferating preadipocytes. Primary preadipocytes isolated using this protocol differentiate rapidly upon reaching confluence and become fully mature in 4-5 days, a temporal window that accurately reflects the appearance of developed fat pads in newborn mice. Primary cultures prepared using this strategy can be expanded and studied with high reproducibility, making them suitable for genetic and phenotypic screens and enabling the study of the cell-autonomous adipocyte phenotypes of genetic mouse models. This protocol offers a simple, rapid, and inexpensive approach to study the complexity of adipose tissue in vitro.

This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture.

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white ...

Differentiated Mouse Adipocytes in Primary Culture: A Model of Insulin Resistance

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance contributes to the development of type 2 diabetes (T2D) and other obesity-derived diseases of high prevalence worldwide. Therefore, understanding the mechanisms underlying insulin resistance is of great relevance. Several models have been used to study insulin resistance both in vivo and in vitro; primary adipocytes represent an attractive option to study the mechanisms of insulin resistance and identify molecules that counteract this condition and the molecular targets of insulin-sensitizing drugs. Here, we have established an insulin resistance model using primary adipocytes in culture treated with tumor necrosis factor-&#945; (TNF-&#945;).
Adipocyte precursor cells (APCs), isolated from collagenase-digested mouse subcutaneous adipose tissue by magnetic cell separation technology, are differentiated into primary adipocytes. Insulin resistance is then induced by treatment with TNF-&#945;, a proinflammatory cytokine that reduces the tyrosine phosphorylation/activation of members of the insulin signaling cascade. Decreased phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS-1), and protein kinase B (AKT) are quantified by western blot. This method provides an excellent tool to study the mechanisms mediating insulin resistance in adipose tissue.

This protocol describes the isolation of mouse preadipocytes from subcutaneous fat, their differentiation into mature adipocytes, and the induction of insulin resistance. Insulin action is evaluated by the phosphorylation/activation of members of the insulin signaling pathway through western blot. This method allows direct determination of insulin resistance/sensitivity in primary adipocytes.

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance ...

Culturing Preadipocytes Isolated from Murine Perivascular Adipose Tissue

Isolation, Culture, and Adipogenic Induction of Stromal Vascular Fraction-derived Preadipocytes from Mouse Periaortic Adipose Tissue

Perivascular adipose tissue (PVAT) is an adipose tissue depot that surrounds blood vessels and exhibits the phenotypes of white, beige, and brown adipocytes. Recent discoveries have shed light on the central role of PVAT in regulating vascular homeostasis and participating in the pathogenesis of cardiovascular diseases. A comprehensive understanding of PVAT properties and regulation is of great importance for the development of future therapies. Primary cultures of periaortic adipocytes are valuable for studying PVAT function and the crosstalk between periaortic adipocytes and vascular cells. This paper presents an economical and feasible protocol for the isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue, which can be useful for modeling adipogenesis or lipogenesis in vitro. The protocol outlines tissue processing and cell differentiation for culturing periaortic adipocytes from young mice. This protocol will provide the technological cornerstone at the bench side for the investigation of PVAT function.

Author Spotlight: The Significance of Isolation, Culture, and Adipogenic Induction of SVF-Derived Preadipocytes from Mouse Perivascular Adipose Tissue 

Here, we describe the isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue, allowing for the study of perivascular adipose tissue function and its relationship with vascular cells.

Perivascular adipose tissue (PVAT) is an adipose tissue depot that surrounds blood vessels and exhibits the phenotypes of white, beige, and brown ...

Culturing Murine Adipocytes and Explants for Functional and Imaging Studies

Isolation and Culturing of Primary Murine Adipocytes from Lean and Obese Mice

Adipose tissue is primarily composed of mature, lipid-laden adipocytes by volume. These postmitotic cells play a critical role in energy storage and mobilization, thermoregulation, and the secretion of endocrine factors. The expansion of white adipose tissue due to caloric imbalance results in both the enlargement of existing adipocytes and the generation of additional adipocytes from adipocyte progenitor cells. Obesity-driven changes to white adipose tissue, including those affecting adipocytes, are associated with numerous comorbidities, such as type 2 diabetes and 13 types of cancer. A significant barrier to studying how adipocytes contribute to disease is the inability to readily isolate and culture mature adipocytes. This article describes a protocol to isolate murine lean and obese adipocytes from the subcutaneous and visceral fat depots of male and female C57BL/6 mice. The protocol details how isolated primary adipocytes can be cultured in a membrane adipocyte aggregate system for up to 2 weeks, facilitating their functional analysis in co-culture experiments, lipolysis assays, or through the collection of conditioned media containing adipocyte-secreted factors. Additionally, the protocol outlines methods for culturing adipose tissue explants in basement membrane matrix domes and imaging primary isolated adipocytes. Importantly, this approach can be integrated with existing protocols for the isolation of adipose tissue-resident adipocyte&#160;progenitor cells using fluorescence-activated cell sorting (FACS). Together, these protocols provide researchers with tools to functionally study adipocytes, adipocyte progenitor cells, and whole adipose tissue from lean and obese, male and female mice.

The size and fragility of mature adipocytes have limited the techniques and tools available for their study and isolation. Here, a protocol is described for the isolation of mature murine adipocytes, which can be easily adapted for different adipose depots and mouse diets.

Adipose tissue is primarily composed of mature, lipid-laden adipocytes by volume. These postmitotic cells play a critical role in energy storage and ...

Isolating Adipose-Derived Stem Cells from Murine Periaortic Adipose Tissue

Source: Qi, Y., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60691">Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue.</a> J. Vis. Exp. (2020)
This video demonstrates a technique for isolating and culturing fluorescently labeled neural crest-derived adipose-derived stem cells (ADSCs) from murine periaortic arch adipose tissue. Following isolation, the NCADSCs are detected, sorted using fluorescence-activated cell sorting, and subsequently cultured on a plate, promoting cell proliferation.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Isolation of the stromal vascular ...

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

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