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

Summary

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.

Abstract

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.

Introduction

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 musculus^1,2.

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 vitro^3,4. 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 myogenesis^5,6. 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 remodeling^7,8.

Previous studies have demonstrated that spiny mouse extracellular matrix has different composition, structure, and properties that support regeneration in comparison to Mus musculus^9,10. In muscle and heart injuries, FAPs have been noted to contribute to superior healing in spiny mouse^11,12,13. 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 musculus^14,15, 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'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-TGFβ1 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).

Protocol

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 °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

1. Prepare 1x Phosphate Buffer Saline (PBS).
2. Prepare PBS-EDTA by mixing 4 mL of 0.5 M EDTA (2 mM) and 996 mL of 1x PBS, pH = 7.4.
3. Prepare 250 mM CaCl₂ by adding 2.7745 g of CaCl₂ to 100 mL of nuclease-free water.
4. Prepare digestion buffer: Mix 50 µg/mL liberase, 2.5 mM CaCl₂, and 5% fetal bovine serum (FBS) in DMEM/F12.
5. Prepare FACS buffer by mixing 488 mL of 1x PBS, 2 mL of 0.5M EDTA, and 10 mL of FBS.
6. Prepare Viability buffer by mixing FACS buffer with propidium iodide (PI; 1 µg/mL).
7. Prepare Basic media by mixing DMEM/F12 with 5% FBS, and 1% (v/v) pen/strep.
8. Prepare Proliferation media by mixing DMEM/F12 with 10% FBS, 1% (v/v) pen/strep and 2.5 ng/mL bFGF.
9. Prepare Fibrogenic media by mixing Basic media and 10 ng/mL rh-TGFβ1,
10. Prepare Adipogenic media (1x) by mixing DMEM/F12 with 1% pen/strep and a commercially available 10x mouse adipogenic differentiation supplement.
11. Prepare 4% PFA by adding 4% Paraformaldehyde (w/v) to 1x PBS.
12. Prepare 0.3% PBS-Triton by mixing 498.5 mL of 1x PBS and 1.5 mL of TritonX-100.
13. Prepare Donkey blocking buffer by mixing 5% (v/v) Donkey serum, 1% BSA (w/v), and 0.3% PBS-Triton.
14. Prepare Donkey + MOM blocking buffer by mixing 2.5 mL of Donkey blocking buffer and 2 drops of MOM block.
15. Prepare DAPI staining solution by mixing 600 nM of 4′,6-diamidino-2-phenylindole (DAPI) in 1x PBS.

2. Tissue collection

1. 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.
2. Place the mouse on the dissection stage in the supine position, and thoroughly spray with 70% ethanol to prevent contamination with mouse fur.
3. 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.
4. 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.
5. 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.
6. 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.
7. Repeat step 2.6 for the other leg.

3. Tissue digestion

1. 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.
2. Mince the tissue with tissue scissors on the lid of the Petri dish into smaller than 1 mm³ pieces.
3. Add minced tissue to their respective digestion tubes. Vortex for 2 s at low speed to mix.
4. Incubate at 37 °C with gentle rotation and shaking.
5. 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.
6. 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 °C with gentle rotation and shaking.
7. 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.
8. Stop the digestion after 60 min by quenching the digestion buffer with ice-cold FACS buffer.
9. Filter the digestion suspension and supernatant through a 40 µm cell strainer on top of 50 mL centrifuge tubes. Top up to 40 mL with FACS buffer.
10. Centrifuge the cell suspension at 500 x g for 8 min at 4 °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.
11. Centrifuge at 500 x g for 5 min at 4 °C. Decant the supernatant.
12. Resuspend in 0.5 mL of FACS buffer, pipetting up and down to ensure cells are evenly dispersed.
13. Filter the suspension through a 40 µm filter on 5 mL polystyrene round-bottom tubes with a cell-strainer cap.

4. Staining for Fluorescence-activated cell sorting (FACS)

1. For single color and fluorescence-minus-one (FMO) controls, prepare 30 µL 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.
2. Prepare single colors and FMO controls for muscle and heart separately.
3. Transfer 30 µL of single color and FMO control staining buffer to a 96-well plate. Top up with 20 µL of FACS buffer.
4. 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.
5. Add 10 µL of the cell suspension to single color and FMO control wells. Add staining buffer to the rest of the cell suspension and resuspend.
6. Incubate at 4 °C, protected from light for 30 min.
7. Top up the wells with 100 µL of FACS buffer, and the sample tube with 2 mL of FACS buffer.
8. Centrifuge at 500 x g for 5 min at 4 °C and remove the supernatant.
9. Repeat steps 4.7 and 4.8.
10. Resuspend the cell pellet in the viability buffer. 100 µL for single color and FMO controls, 1 mL for FACS sorting samples.
11. Prepare the collection tubes by adding 300 µL of proliferation media in 1.7 mL centrifuge tubes.

5. Fluorescence-activated cell sorting

1. 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 PDGFRα to gate for FAPs (Figure 2).
2. Sort into collection tubes previously prepared.

6. Tissue culture

1. Centrifuge the collected cells at 800 x g for 10 min at 4 °C.
2. Remove the supernatant with a P1000 pipette. Be careful not to disturb the cell pellet.
3. Resuspend the pellet in culture media (250 µL/well) and seed at 25k cells/well in a 48-well tissue culture plate.
4. Culture in an incubator (37 °C, 5% CO₂). Change media every 3 days until cells reach 80% confluency.

7. Fibrogenic differentiation

1. 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.
2. Aspirate the culture media.
3. Rinse with 200 µL of 1x DPBS twice.
4. Rinse with 200 µL of basic media once.
5. Add 250 µL of fibrogenic or basic media to the respective wells.
6. Return the cells to the incubator (37 °C, 5% CO₂).
7. Stop the differentiation after 48 h.

8. Adipogenic differentiation

1. 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.
2. Aspirate the culture media.
3. Rinse with 200 µL of 1x DPBS twice.
4. Rinse with 200 µL of basic media once.
5. Add 400 µL of adipogenic or basic media to respective wells.
6. Return to incubator (37°C, 5% CO₂).
7. Every 2 days, remove 200 µL media with a pipette from the wells and add 200 µL of fresh adipogenic or basic media.
8. Stop differentiation on the 6^th day for skeletal muscle FAPs, and 14^th day for cardiac FAPs.

9. Immunostaining

1. Stop the differentiation assay by aspirating media and rinsing with 250 µL of 1x PBS twice. Perform all washes with 18 G needle attached to 3 mL syringe to avoid cell detachment.
2. Add 250 µL of 4% PFA per well and incubate at room temperature for 7 min.
3. Remove the PFA and rinse 3 times for 5 min each time with 250 µL of 1x PBS.
4. On the last wash, aspirate 1x PBS and add 250 µL of Donkey + MOM blocking buffer.
5. Incubate at room temperature for 60 min.
6. Prepare primary antibody staining solution in Donkey blocking buffer (anti-SMA and anti-perilipin) for a final volume of 100 µL per well.
7. Aspirate the staining wells. Leave Donkey + MOM blocking buffer in control well if applicable.
8. Add primary antibody staining solution in the respective wells.
9. Incubate at 4 °C overnight.
10. Next day, wash 3 times for 5 min each time with 1x PBS.
11. Prepare secondary antibody staining solution in Donkey blocking buffer with a final volume of 100 µL per well.
12. Aspirate the last wash and add secondary antibody staining solution in all wells.
13. Incubate at room temperature for 45 min (samples should be protected from light from this point forward).
14. Wash 3 times for 5 min each time with 1x PBS.
15. Aspirate the last wash and add 250 µL of DAPI staining solution.
16. Incubate at room temperature for 5 min.
17. Remove the DAPI staining solution and wash once for 5 min with 1x PBS.
18. Add 100 µL of Fluoromount-G in each well.
19. Seal the plate with the lid and paraffin film to minimize evaporation. Store at 4 °C, protected from the light until imaging.
20. 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.

Results

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).

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. Please click here to view a larger version of this figure.

Figure 2: Representative FACS plots for skeletal muscle (top) and cardiac (bottom) FAP isolation. Please click here to view a larger version of this figure.

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 µm. Please click here to view a larger version of this figure.

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 µm. Please click here to view a larger version of this figure.

Figure 5: Brightfield images of spiny mouse skeletal muscle (A) and heart (B) FAPs after 5 days of culture, before differentiation. Scale bar = 360 µm. Please click here to view a larger version of this figure.

Controls	CD31 SC	PDGFRα SC	CD31 FMO	PDGFRα FMO	Sample
CD31 antibody	+	-	-	+	+
PDGFRα antibody	-	+	+	-	+

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.

Discussion

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 PDGFRα 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 context^18,19,20. 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 models^5,7. Characterizing spiny mouse FAPs may elucidate the mechanism behind their remarkable regenerative capacity and pave the way toward novel strategies in regenerative medicine.

Materials

List of materials used in this article

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