We would like to thank the Flow cytometry Core Facilities at the University of Ottawa and the Keenan Research Centre for Biomedical Sciences (KRC), St Michaels Hospital Unity Health Toronto for their expertise and guidance in optimization of the flow cytometry/FACS protocol presented in this manuscript. This work was funded by Medicine by Design New Ideas 2018 Fund (MbDNI-2018-01) to JB.

The authors have no conflicts to disclose.

An optimized, validated FAPs isolation protocol for rat muscle is essential for researchers who wish to study injury models that are not feasible in the mouse for biologic or technical reasons. For example, mice are not an optimal animal model in which to study chronic local, or neurodegenerative injuries such as long-term denervation. Biologically, the short lifespan and rapid aging of mice make it difficult to accurately delineate the muscle sequalae due to denervation from the confounding factor of aging. From a techn...

Fibro-adipogenic progenitor cells (FAPs) are a population of resident multipotent progenitor cells in skeletal muscle that play a critical role in muscle homeostasis, repair, and regeneration, and conversely, also mediate pathologic responses to muscle injury. As the name suggests, FAPs were originally identified as a progenitor population with the potential to differentiate into fibroblasts and adipocytes1&#160;and were purported to be the key mediators of fibro-fatty infiltration of skeletal muscle in chronic injury and disease. Further study revealed that FAPs are additionally capable of osteogenesis and chondrogenesis2,3,4. Thus, they are more broadly notated in the literature as mesenchymal or stromal progenitors3,5,6,7,8. In acute skeletal muscle injury, FAPs indirectly aid in regenerative myogenesis by transiently proliferating to provide a favorable environment for activated muscle satellite cells and their downstream myogenic progenitor (MPs) counterparts1,9,10. In parallel with successful regeneration, FAPs undergo apoptosis, returning their numbers to baseline levels1,9,10,11. In contrast, in chronic muscle injury, FAPs override pro-apoptotic signals, which results in their persistence9,10,11&#160;and abnormal muscle repair.
In vivo studies evaluating the cellular and molecular mechanisms by which FAPs mediate muscle responses have utilized murine animal models to date1,7,9,10,11,12,13,14. While genetically engineered mice are powerful tools for&#160;use in these analyses, the small size of the animal limits tissue availability for study in long-term localized injury models where muscle atrophy can be profound, such as traumatic denervation. Furthermore, measurement of muscle strength and physical function requires ex vivo or in situ measurements that necessitate termination of the mouse, or in vivo methods that require surgery and/or a general anaesthetic to permit evaluation of muscle contractile performance15,16,17,18,19,20. In rats, well validated and globally utilized muscle functional analyses, in addition to analyses for more complex motor behaviors such as gait analysis (e.g., Sciatic Function Index, CatWalk analysis) exist and are performed in awake and spontaneously moving animals21,22,23,24. This additionally optimizes the principles of minimal morbidity in animal experimentation, and numbers of research animals used. The rat thereby provides the FAPs investigator the added flexibility of greater injured muscle volume for protein and cellular analyses and the ability to undertake serial assessments of muscle complex static and dynamic functional activity and behaviors, in the alert animal.
FAPs have primarily been identified and isolated from whole muscle samples using flow cytometry and Fluorescence-activated cell sorting (FACS) respectively. These are laser-based assays that are able to identify multiple specific cell populations based on characteristic features such as size, granularity, and a specific combination of cell surface or intracellular markers25. This is highly advantageous in the study of an organ system such as skeletal muscle, as homeostasis and regeneration are&#160;complex, multifactorial processes coordinated by a plethora of cell types. A seminal study identified FAPs, as well as MPs, using flow cytometric methods in mouse skeletal muscle1. They demonstrated that FAPs are mesenchymal in nature, as they lacked surface antigens specific to cells from endothelial (CD31), hematopoietic (CD45), or myogenic (Integrin-&#945;7 [ITGA7]) origins, but expressed the mesenchymal stem cell marker Sca-1 (Stem cell antigen 1)1&#160;and differentiated into fibrogenic and adipogenic cells in culture. Other studies demonstrated successful isolation of mesenchymal progenitors in muscle based on the expression of an alternative stem cell marker, platelet-derived growth factor receptor alpha (PDGFR&#945;)2,7,8&#160;and further analysis revealed these likely to be the same cell population as FAPs3. FAPs are now commonly identified in flow cytometry using either Sca-1 or PDGFR&#945; as a positive selection marker1,9,10,11,12,13,14,26,27,28,29,30,31. The use of PDGFR&#945; is preferential for human tissue&#160;however, as a direct human homologue of murine Sca-1 has yet to be identified32. In addition, other cell surface proteins have been reported as markers of MPs (e.g., VCAM-1), providing a potential alternative to ITGA7 as an indicator of cells of myogenic lineage during FAPs isolation33.
While flow cytometry/FACS is a powerful methodology for studying the role and pathogenic potential of FAPs in skeletal muscle1,9,10,11,13,29, it is limited technically by the specificity and optimization of its required reagents. Since flow cytometric identification and isolation of FAPs has been developed and conducted in mouse animal models1,9,10,11,29, this poses challenges for researchers who wish to study FAPs in other model organisms. Many factors - such as optimal tissue size to be processed, as well as reagent and/or antibody specificity and availability - differ depending on the species used.
In addition to the technical barriers to studying FAPs in a novel animal model, they have largely been studied in an acute, toxic setting - usually via intramuscular chemical injection or cardiotoxin. Evaluation of the long-term dynamics of FAPs is limited primarily to assessment in Duchenne&#39;s muscular dystrophy, using the mdx mouse model9,10,11, and models of combination muscle injury such as massive rotator cuff tear where concurrent tendon transection and denervation is performed on shoulder musculature26,27,28. The response of FAPs to the sole insult of chronic traumatic denervation, a common occurrence in work-place accidents in heavy industry, agriculture, and in birth traumas (brachial plexus injury)34,35,36,37&#160;with significant morbidity, has not been as well characterized, often limited to a short-term time frame11,38.
We describe a method for identifying and isolating FAPs and MPs from healthy as well as severely atrophic and fibrotic skeletal muscle in the rat. First, identification of CD31-/CD45-/Sca-1+/VCAM-1- FAPs and CD31-/CD45-/Sca-1-/VCAM-1+ MPs using a tissue digestion and flow cytometry staining protocol is demonstrated and subsequent validation of our findings is performed through culture and immunocytochemical staining of FACS-isolated cells. Using this method, we also report a novel FAPs time-course in a long-term isolated denervation injury model in the rat.

<table><tbody><tr><td>5 mL Polypropylene Round-Bottom Tube</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>10 cm cell culture dishes</td><td>Sarstedt</td><td>83.3902</td><td></td></tr><tr><td>12-well cell culture plate</td><td>ThermoFisher</td><td>130185</td><td></td></tr><tr><td>12 mm glass coverslips, No.2</td><td>VWR</td><td>89015-724</td><td></td></tr><tr><td>10 mL Syringe</td><td>Beckton Dickenson</td><td>302995</td><td></td></tr><tr><td>15 mL centrifuge tubes</td><td>FroggaBio</td><td>91014</td><td></td></tr><tr><td>20 gauge needle</td><td>Beckton Dickenson</td><td>305176</td><td></td></tr><tr><td>25mL Serological pipette</td><td>Sarstedt</td><td>86.1685.001</td><td></td></tr><tr><td>40&amp;micro;m cell strainer</td><td>Fisher Scientific</td><td>22363547</td><td></td></tr><tr><td>50mL centrifuge tubes</td><td>FroggaBio</td><td>TB50</td><td></td></tr><tr><td>AbC Total Antibody Compensation Beads&amp;nbsp;</td><td>ThermoFisher</td><td>A10497</td><td></td></tr><tr><td>Ammonium Chloride, Reagent Grade</td><td>Bioshop</td><td>AMC303.500</td><td></td></tr><tr><td>APC Conjugation Kit, 50-100&amp;micro;g</td><td>Biotium</td><td>92307</td><td></td></tr><tr><td>Aquatex Aqueous Mounting Medium</td><td>Merck</td><td>108562</td><td></td></tr><tr><td>Biolaminin 411 LN</td><td>Biolamina</td><td>LN411</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Bioshop</td><td>ALB001</td><td></td></tr><tr><td>Calcium Chloride</td><td>Bioshop</td><td>CCL444.500</td><td></td></tr><tr><td>Collagenase Type II</td><td>Gibco</td><td>17101015</td><td></td></tr><tr><td>CountBright Plus Absolute Counting Beads</td><td>ThermoFisher</td><td>C36995</td><td></td></tr><tr><td>Dexamethasone</td><td>Millipore Sigma</td><td>D4902</td><td></td></tr><tr><td>Dispase</td><td>Gibco</td><td>17105041</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM) (1X)</td><td>Gibco</td><td>11995-065</td><td>(+)4.5 g/L D-Glucose&lt;br/&gt; (+)L-Glutamine&lt;br/&gt; (+)110 mg/L Sodium Pyruvate </td></tr><tr><td>EDTA</td><td>FisherScientific</td><td>S311</td><td></td></tr><tr><td>FACSClean Solution</td><td>Beckton Dickenson</td><td>340345</td><td></td></tr><tr><td>FACSDiva Software</td><td>Beckton Dickenson</td><td>--</td><td></td></tr><tr><td>FACSRinse Solution</td><td>Beckton Dickenson</td><td>340346</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma</td><td>F1051</td><td></td></tr><tr><td>Flow Cytometry Sheath Fluid</td><td>Beckton Dickenson</td><td>342003</td><td></td></tr><tr><td>FlowJo Software</td><td>Beckton Dickenson</td><td>--</td><td></td></tr><tr><td>Fluorescent Mounting Medium</td><td>Dako</td><td>S302380-2</td><td></td></tr><tr><td>Goat anti-mouse Alexa Fluor 555 secondary antibody</td><td>Invitrogen</td><td>A21424</td><td></td></tr><tr><td>Goat anti-rabbit Alexa Fluor 488 secondary antibody</td><td>Invitrogen</td><td>A11008</td><td></td></tr><tr><td>Goat anti-rabbit Alexa Fluor 555 secondary antibody</td><td>Invitrogen</td><td>A21429</td><td></td></tr><tr><td>Goat Serum</td><td>Gibco</td><td>16210-064</td><td></td></tr><tr><td>Ham&#039;s F10 Media</td><td>ThermoFisher&amp;nbsp;</td><td>11550043</td><td>(+) Phenol Red&lt;br/&gt; (+) L-Glutamine&lt;br/&gt; (-) HEPES</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution (HBSS) (1X)</td><td>Multicell</td><td>311-513-CL</td><td></td></tr><tr><td>Heat Inactivated Horse Serum</td><td>Gibco</td><td>26050-088</td><td></td></tr><tr><td>Hemocytometer</td><td>Reichert</td><td>N/A</td><td></td></tr><tr><td>HEPES, minimum 99.5% titration</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>Horse Serum</td><td>ThermoFisher</td><td>16050130</td><td></td></tr><tr><td>Human Transforming Growth Factor &amp;beta;1 (hTGF-&amp;beta;1)</td><td>Cell Signaling</td><td>8915LF</td><td></td></tr><tr><td>Humulin R</td><td>Lilly</td><td>HI0210</td><td></td></tr><tr><td>IBMX</td><td>Millipore Sigma</td><td>I5879</td><td>Also known as 3-Isobutyl-1-methylxanthine</td></tr><tr><td>Isopropanol</td><td>Sigma</td><td>I9516</td><td>Also known as 2-propanol</td></tr><tr><td>Lewis Rat, Female</td><td>Charles River Kingston</td><td>004 (Strain Code)</td><td>200-250 grams used</td></tr><tr><td>LSRFortessa X-20 Benchtop Cytometer</td><td>Beckton Dickenson</td><td>--</td><td></td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>EP-5417R</td><td></td></tr><tr><td>MoFlo XDP Cell Sorter</td><td>Beckman Coulter</td><td>--</td><td></td></tr><tr><td>Mouse Anti-CD31::FITC Antibody</td><td>Abcam</td><td>ab33858</td><td>Clone TLD-3A12</td></tr><tr><td>Mouse Anti-CD45::FITC Antibody</td><td>Biolegend</td><td>202205</td><td>Clone OX-1</td></tr><tr><td>Mouse Anti-CD106::PE Antibody</td><td>Biolegend</td><td>200403</td><td>Also known as VCAM-1</td></tr><tr><td>Mouse Anti-MHC Antibody</td><td>Developmental Studies Hybridoma Bank (DSHB)</td><td>N/A</td><td>Also known as MF20</td></tr><tr><td>Mouse Anti-Pax7 Antibody</td><td>Developmental Studies Hybridoma Bank (DSHB)</td><td>N/A</td><td></td></tr><tr><td>Neutral Buffered Formalin, 10 %</td><td>Sigma</td><td>HT501128</td><td></td></tr><tr><td>Oil Red O</td><td>Millipore Sigma</td><td>O0625</td><td></td></tr><tr><td>PE-Cy7 Conjugation Kit</td><td>Abcam</td><td>ab102903</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma&amp;nbsp;</td><td>P4333</td><td></td></tr><tr><td>Phosphate Buffered Saline, pH 7.4 (1X)</td><td>Gibco</td><td>10010-023</td><td>(-)Calcium Chloride&lt;br/&gt; (-)Magnesium Chloride </td></tr><tr><td>Potassium Bicarbonate, Reagent Grade</td><td>Bioshop</td><td>PBC401.250</td><td></td></tr><tr><td>Rabbit Anti-Fibroblast Specific Protein 1 (FSP-1) Antibody</td><td>Invitrogen</td><td>MA5-32347</td><td>FSP-1 also known as S100A4</td></tr><tr><td>Rabbit Anti-Integrin-a7 Antibody</td><td>Abcam</td><td>ab203254</td><td></td></tr><tr><td>Rabbit Anti-Laminin Antibody</td><td>Sigma</td><td>L9393</td><td></td></tr><tr><td>Rabbit Anti-Perilipin-1 Antibody</td><td>Abcam</td><td>ab3526</td><td></td></tr><tr><td>Rabbit Anti-Sca-1 Antibody</td><td>Millipore Sigma</td><td>AB4336</td><td></td></tr><tr><td>Rabbit Recombinant Anti-Collagen Type I Antibody</td><td>Abcam</td><td>ab260043</td><td>Also known as Col1a1</td></tr><tr><td>Rabbit Recombinant Anti-PDGFR Alpha Antibody</td><td>Abcam</td><td>ab203491</td><td></td></tr><tr><td>Recombinant Human FGF-basic</td><td>Gibco</td><td>PHG0266</td><td></td></tr><tr><td>Sodium Azide</td><td>Sigma</td><td>S2002</td><td></td></tr><tr><td>Triton-X-100</td><td>Fisher Scientific</td><td>BP151</td><td></td></tr><tr><td>Troglitazone</td><td>Millipore Sigma</td><td>T2573</td><td></td></tr><tr><td>Tween-20</td><td>Bioshop</td><td>TWN510</td><td></td></tr></tbody></table>

identification, isolation, and characterization of fibro-adipogenic progenitors (faps) and myogenic progenitors (mps) in skeletal muscle in the rat

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in muscle homeostasis, injury, and repair. Current protocols for FAPs identification and isolation use flow cytometry/fluorescence-activated cell sorting (FACS) and studies evaluating their function in vivo to date have been undertaken exclusively in mice. The larger inherent size of the rat allows for a more comprehensive analysis of FAPs in skeletal muscle injury models, especially in severely atrophic muscle or when investigators require substantial tissue mass to conduct multiple downstream assays. The rat additionally provides a larger selection of muscle functional assays that do not require animal sedation or sacrifice, thus minimizing morbidity and animal use by enabling serial assessments. The flow cytometry/FACS protocols optimized for mice are species specific, notably restricted by the characteristics of commercially available antibodies. They have not been optimized for separating FAPs from rat or highly fibrotic muscle. A flow cytometry/FACS protocol for the identification and isolation of FAPs and MPs from both healthy and denervated rat skeletal muscle was developed, relying on the differential expression of surface markers CD31, CD45, Sca-1, and VCAM-1. As rat-specific, flow cytometry-validated primary antibodies are severely limited, in-house conjugation of the antibody targeting Sca-1 was performed. Using this protocol, successful Sca-1 conjugation was confirmed, and flow cytometric identification of FAPs and MPs was validated by cell culture and immunostaining of FACS-isolated FAPs and MPs. Finally, we report a novel FAPs time-course in a prolonged (14 week) rat denervation model. This method provides the investigators the ability to study FAPs in a novel animal model.

Identifying FAPs and MPs via flow cytometry using a novel antibody panel including Sca-1 and VCAM-1 
The gating strategy for identifying FAPs in rat muscle is based upon flow cytometry protocols in the mouse29, which gate on CD31 (endothelial) and CD45 (hematopoietic) positive cells (termed the lineage [Lin]) and examines the fluorescent profile of FAPs marker Sca-1 and MPs marker ITGA7 from the linage-negative (Lin-) populat...

Watch this Scientific Journal Video about Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors FAPs and Myogenic Progenitors MPs in Skeletal Muscle in the Rat at JoVE.com

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors FAPs and Myogenic Progenitors MPs in Skeletal Muscle in the Rat

This protocol outlines a method to isolate Fibro-adipogenic progenitors (FAPs) and myogenic progenitors (MPs) from rat skeletal muscle. Utilization of the rat in muscle injury models provides increased tissue availability from atrophic muscle for the analysis and a larger repertoire of validated methods to assess muscle strength and gait in free-moving animals.

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors (FAPs) and Myogenic Progenitors (MPs) in Skeletal Muscle in the Rat

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in ...

identification-isolation-characterization-fibro-adipogenic

Research

JoVE Journal

Biology

5.9K Views.  St Michaels Hospital, Unity Health Toronto. Aangezien FAP's mediatoren zijn van spierregeneratie en pathologische fibrose, maakt ons protocol het mogelijk om de FAP-dynamiek na spierletsel te karakteriseren en FAP's te isoleren voor in vitro of ex vivo studies. Tot op heden zijn studies uitsluitend uitgevoerd op FAPs geïsoleerd van muizen. Ons protocol maakt een effectieve isolatie van FAPs van de grotere rat mogelijk, waardoor er veel meer weefsel beschikbaar is voor downstream-assays.Langdurige weefselverwerkingstijd kan de l...

Video: Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors FAPs and Myogenic Progenitors MPs in Skeletal Muscle in the Rat

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Developmental Biology

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

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 ...

A Guide to Examining Intramuscular Fat Formation and its Cellular Origin in Skeletal Muscle

Fibro-adipogenic progenitors (FAPs) are mesenchymal stromal cells that play a crucial role during skeletal muscle homeostasis and regeneration. FAPs build and maintain the extracellular matrix that acts as a molecular myofiber scaffold. In addition, FAPs are indispensable for myofiber regeneration as they secrete a multitude of beneficial factors sensed by the muscle stem cells (MuSCs). In diseased states, however, FAPs are the cellular origin of intramuscular fat and fibrotic scar tissue. This fatty fibrosis is a hallmark of sarcopenia and neuromuscular diseases, such as Duchenne Muscular Dystrophy. One significant barrier in determining why and how FAPs differentiate into intramuscular fat is effective preservation and subsequent visualization of adipocytes, especially in frozen tissue sections. Conventional methods of skeletal muscle tissue processing, such as snap-freezing, do not properly preserve the morphology of individual adipocytes, thereby preventing accurate visualization and quantification. To overcome this hurdle, a rigorous protocol was developed that preserves adipocyte morphology in skeletal muscle sections allowing visualization, imaging, and quantification of intramuscular fat. The protocol also outlines how to process a portion of muscle tissue for RT-qPCR, enabling users to confirm observed changes in fat formation by viewing differences in the expression of adipogenic genes. Additionally, it can be adapted to visualize adipocytes by whole-mount immunofluorescence of muscle samples. Finally, this protocol outlines how to perform genetic lineage tracing of Pdgfr&#945;-expressing FAPs to study the adipogenic conversion of FAPs. This protocol consistently yields high-resolution and morphologically accurate immunofluorescent images of adipocytes, along with confirmation by RT-qPCR, allowing for robust, rigorous, and reproducible visualization and quantification of intramuscular fat. Together, the analysis pipeline described here is the first step to improving our understanding of how FAPs differentiate into intramuscular fat, and provides a framework to validate novel interventions to prevent fat formation.

The replacement of healthy muscle tissue with intramuscular fat is a prominent feature of human diseases and conditions. This protocol outlines how to visualize, image, and quantify intramuscular fat, allowing the rigorous study of the mechanisms underlying intramuscular fat formation.

Fibro-adipogenic progenitors (FAPs) are mesenchymal stromal cells that play a crucial role during skeletal muscle homeostasis and regeneration. FAPs build ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response. The resulting increase in the number of circulating red cells improves tissue oxygen delivery. An efficient erythropoietic stress response is therefore critical to the survival and recovery from physiological and pathological conditions such as high altitude, anemia, hemorrhage, chemotherapy or stem cell transplantation. 
The mouse is a key model for the study of erythropoiesis and its stress response. Mouse definitive (adult-type) erythropoiesis takes place in the fetal liver between embryonic days 12.5 and 15.5, in the neonatal spleen, and in adult spleen and bone marrow. Classical methods of identifying erythroid progenitors in tissue rely on the ability of these cells to give rise to red cell colonies when plated in Epo-containing semi-solid media. Their erythroid precursor progeny are identified based on morphological criteria. Neither of these classical methods allow access to large numbers of differentiation-stage-specific erythroid cells for molecular study. Here we present a flow-cytometric method of identifying and studying differentiation-stage-specific erythroid progenitors and precursors, directly in the context of freshly isolated mouse tissue. The assay relies on the cell-surface markers CD71, Ter119, and on the flow-cytometric 'forward-scatter' parameter, which is a function of cell size. The CD71/Ter119 assay can be used to study erythroid progenitors during their response to erythropoietic stress in vivo, for example, in anemic mice or mice housed in low oxygen conditions. It may also be used to study erythroid progenitors directly in the tissues of genetically modified adult mice or embryos, in order to assess the specific role of the modified molecular pathway in erythropoiesis.

A flow-cytometric method for identification and molecular analysis of differentiation-stage-specific murine erythroid progenitors and precursors, directly in freshly &#x2013;harvested mouse bone marrow, spleen or fetal liver. The assay relies on cell-surface markers CD71, Ter119, and cell size.

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of ...

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

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

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 ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Adipose Tissue-Derived Stem Cell Isolation and Characterization

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

Author Spotlight: Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...

Single-Cell CyTOF Analysis of Myogenic Progenitors in Muscle Regeneration

Identification and Analysis of Myogenic Progenitors In Vivo During Acute Skeletal Muscle Injury by High-Dimensional Single-Cell Mass Cytometry

Skeletal muscle regeneration is a dynamic process driven by adult muscle stem cells and their progeny. Mostly quiescent at a steady state, adult muscle stem cells become activated upon muscle injury. Following activation, they proliferate, and most of their progeny differentiate to generate fusion-competent muscle cells while the remaining self-renews to replenish the stem cell pool. While the identity of muscle stem cells was defined more than a decade ago, based on the co-expression of cell surface markers, myogenic progenitors were identified only recently using high-dimensional single-cell approaches. Here, we present a single-cell mass cytometry (cytometry by time of flight [CyTOF]) method to analyze stem cells and progenitor cells in acute muscle injury to resolve the cellular and molecular dynamics that unfold during muscle regeneration. This approach is based on the simultaneous detection of novel cell surface markers and key myogenic transcription factors whose dynamic expression enables the identification of activated stem cells and progenitor cell populations that represent landmarks of myogenesis. Importantly, a sorting strategy based on detecting cell surface markers CD9 and CD104 is described, enabling prospective isolation of muscle stem and progenitor cells using fluorescence-activated cell sorting (FACS) for in-depth studies of their function. Muscle progenitor cells provide a critical missing link to study the control of muscle stem cell fate, identify novel therapeutic targets for muscle diseases, and develop cell therapy applications for regenerative medicine. The approach presented here can be applied to study muscle stem and progenitor cells in vivo in response to perturbations, such as pharmacological interventions targeting specific signaling pathways. It can also be used to investigate the dynamics of muscle stem and progenitor cells in animal models of muscle diseases, advancing our understanding of stem cell diseases and accelerating the development of therapies.

Author Spotlight: Investigating Cellular and Molecular Dynamics During Muscle Regeneration Using Cutting-Edge Single-Cell Technologies

The protocol presented here enables the identification and high-dimensional analysis of muscle stem and progenitor cells by single-cell mass cytometry and their purification by FACS for in-depth studies of their function. This approach can be applied to study regeneration dynamics in disease models and test the efficacy of pharmacological interventions.

Skeletal muscle regeneration is a dynamic process driven by adult muscle stem cells and their progeny. Mostly quiescent at a steady state, adult muscle ...

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors (FAPs) and Myogenic Progenitors (MPs) in Skeletal Muscle in the Rat

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