digestion of mouse skeletal and cardiac muscles for fluorescence-activated cell sorting and culture of fibro-adipogenic progenitors (faps)

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

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).

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.

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

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

digestion-mouse-skeletal-cardiac-muscles-for-fluorescence-activated

Research

JoVE Journal

Developmental Biology

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.

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

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

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

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

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

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