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Cell sorting of various cell types from mouse and human skeletal muscle
Methods xxx (2017) xxx–xxx
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Cell sorting of various cell types from mouse and human skeletal muscle Claire Latroche a, Michèle Weiss-Gayet b, Cyril Gitiaux a, Bénédicte Chazaud b,⇑ a b
Institut Cochin, INSERM U1016, CNRS UMR8104, Université Paris Descartes, PRES Sorbonne-Paris-Cité, Paris, France Institut NeuroMyoGène, Univ Lyon, Université Claude Bernard Lyon 1, INSERM U1217, CNRS UMR5310, Lyon, France
a r t i c l e
i n f o
Article history: Received 30 June 2017 Received in revised form 11 December 2017 Accepted 15 December 2017 Available online xxxx Keywords: Skeletal muscle regeneration Cell sorting Myogenic cells Fibroblasts Macrophages Endothelial cells
a b s t r a c t Muscle stem cells or satellite cells are required for skeletal muscle regeneration. It has been shown that the satellite cell microenvironment, including neighboring cells such as endothelial cells, macrophages or fibroblasts are essential for complete and efficient regeneration. A deficient behavior of these cells compromises regeneration. Therefore, there is a strong interest in understanding the cellular and molecular interactions at work between these cell types during muscle regeneration. Fluorescence-activated cell sorting allows to isolate these four cell types at different time points of regeneration, for further high throughput or behavioral experiments. We present here a method for the concomitant isolation of 4 cell types present in the regenerating skeletal muscle: muscle stem cells, endothelial cells, fibro-adipogenic precursor cells and macrophages. Ó 2017 Elsevier Inc. All rights reserved.
1. Introduction Normal adult skeletal muscle regenerates ad integrum after injury. In experimental injury models in mouse, muscle tissue undergoes necrosis followed by rapid infiltration of immune cells. Few days later (depending on the type and extent of the injury), new myofibers are formed. After about one month, regenerating muscle is similar to uninjured muscle except that the myofibers exhibit central nuclei. Satellite cells (SCs) are the principal contributors for the regeneration of skeletal muscle. Upon myofiber damage, quiescent SCs become activated, divide, and give rise to a population of transient amplifying myogenic precursor cells (skeletal myoblasts). Later on, most of these myogenic precursors exit the cell cycle, lose Pax7 expression, differentiate and fuse with the host fibers or between them to form new myofibers. A subset of myogenic precursor does not differentiate and self-renews to replenish the pool of SCs [1]. While SCs are indispensable for skeletal muscle regeneration [2,3], other cell types, such as fibroblasts, endothelial cells and immune cells, among which macrophages, have been shown to play important roles in this process. Indeed, these cells establish specific interactions with SCs to ensure efficient muscle regeneration. However, the cell dynamics are highly complex and occur with specific temporal and spatial kinetics. ⇑ Corresponding author at: Institut NeuroMyoGène, Université Claude Bernard Lyon 1, 8 Avenue Rockfeller, 69003 Lyon, France. E-mail address: [email protected] (B. Chazaud).
Immune cells invade the muscle early after injury, first as neutrophils, then as macrophages. Macrophages serve as key effectors in the muscle stem cell niche to guide SCs through the regeneration process through direct interactions with myogenic cells. They first invade the muscle as inflammatory macrophages (coming from inflammatory circulating monocytes) and specifically stimulate myogenic precursor proliferation and trigger Fibro-Adipogenic Precursor (FAP) apoptosis (see below). Then, upon the phagocytosis of necrotic muscle debris, they switch their inflammatory status into anti-inflammatory cells, that stimulate myogenic differentiation and promote myofiber growth [4–6]. Skeletal muscle is laced with a dense microvasculature and most quiescent SCs are located close to capillaries. Vascular cells also coordinate both the acute SC response and the late stages of muscle regeneration when the tissue returns to homeostasis. Endothelial cells (ECs) predominantly exhibit pro-myogenic effects on SCs, through a strong stimulating effect on their differentiation, while peri-endothelial cells, such as smooth muscle cells and fibrogenic cell types, are crucial for the re-entry into quiescence on completion of regeneration. Inversely, SCs and myogenic precursors have been shown to exhibit pro-angiogenic activity [6–8]. Finally, fibroblasts and FAPs are the main source of matrix proteins during muscle regeneration and are required for muscle regeneration [9]. Upon muscle injury, FAPs are activated, rapidly expand and control matrix remodeling, while providing an environment favoring myogenic differentiation [10,11]. While these observations show the existence of specific interactions between SCs and the neighboring cells during muscle
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regeneration, the molecular mechanisms involved in this complex network of communication are still poorly known. In this study, we describe a protocol for isolating pure populations of SCs, macrophages, ECs and FAPs from regenerating mouse skeletal muscle and for isolating SCs and ECS from human muscle samples.
2. Isolation of murine muscle cells
2.2.1. Cell preparation 1. Prior dissection, prepare the digestion medium (collagenase B 10 mg/ml – dispase II 2,4U/ml) in DMEM/F12 serum-free (1 ml/muscle). Heat the medium at 37 °C and filtrate through a 0.22 mm filter. 2. Dissect Tibialis Anterior of both hind limbs. In a Petri dish on ice, roughly discard visible fat, tendons and fascia.
2.1. Induction of muscle regeneration Muscle injury was performed by the injection of cardiotoxin (CTX), a kinase C inhibitor extracted from Anja nigricollis snake venom, that induces depolarization and contraction of muscle cells and destroys the cell membrane structure [12]. CTX injection provides an homogeneous damage in the whole muscle, and the following sequential steps of the regeneration process are well described [13]. Intramuscular injection of CTX (12 mM, 50 ml, Latoxan, France) was performed in the Tibialis Anterior muscle of adult male mice [14]. Adult 6–8 week-old male C57Bl/6 mice were bred and used in compliance with French and European regulations. Principal investigator is licensed for these experiments and the protocols were approved by local Animal Care and Use Committee and the French Ministry of Agriculture. Usually, the time points studied are days 1, 2, 4 and 8 after injury. At day 1 after injury, myofibers are necrotic, while immune cells (neutrophils and circulating monocytes) enter into the damaged muscle. At day 2, macrophages phagocyte the dead myofibers and FAPs are highly expanding while SCs are also in the amplification phase. At day 4 after injury, the regeneration process is visible with the appearance of the new regenerating myofibers indicative of myogenic differentiation. Macrophages are still numerous in supporting this process, while FAPs are remodeling the matrix and angiogenesis takes place. At day 8, muscle has almost recovered its original appearance, with nanofibers characterized by the central location of their nuclei, microvasculature maturing and the almost disappearance of macrophages and FAPs.
2.2. Flow cytometry
Table 2 Reagents. Reagent
Cat. No
Manufacturer
Murine muscle cell isolation DMEM/F12 Collagenase B 10 mg/ml Dispase II 2.4 U/ml Fetal Bovine Serum ACK Buffer 1X PBS Fc block Anti a7 integrin-647 Anti CD34-FITC Anti CD45-PE Anti CD31-eFluor450 Anti Sca1-PerCP-Cy5.5 Anti F4/80 APC-eFluor780 Anti CD140a PE-Cy7 Rat IgG2a Isotype control FITC Rat IgG2b Isotype control PE Rat IgG2b Isotype control eFluor 450 Rat IgG2a Isotype control PerCP-Cy5-5 Rat IgG2a Isotype control APC-eFluor 780 Rat IgG2a Isotype control PE-Cy7
31331-028 11 088 831 001 04 942 078 001 10270-106 10-548E 14190-094 130-059-901 AB0000538 11-0341 12-0451 48-0311 45-5981 47-4801 25-1401 11-4321 12-4031 48-4031 45-4321 47-4321 25-4321
Gibco Roche Roche Gibco Lonza Gibco Miltenyi AB lab eBioscience eBioscience eBioscience eBioscience eBioscience eBioscience eBioscience eBioscience eBioscience eBioscience eBioscience eBioscience
Human muscle cell isolation ECGMV2 Collagenase B 1 mg/ml Dispase II 2.4 U/ml Fetal Bovine Serum 1X PBS Anti CD31-FITC Anti CD56-APC
C-22022 11 088 831 001 04 942 078 001 10270-106 14190-094 11-0319 555518
Mouse IgG1 Isotype Control FITC Mouse IgG1 Isotype Control APC
11-4714 555751
Promocell Roche Roche Gibco Gibco eBioscience BD Pharmingen eBioscience BD Pharmingen
All materials and reagents used are listed in Tables 1 and 2 respectively.
Table 1 Material. Material
Cat. No
Manufacturer
Murine muscle cell isolation Material for mouse dissection (thin forceps, rasor blade or thin scissors, thin sharp scissors) Petri dishes 0.22 mm filters Polypropylene Round Bottom Tube Polystyrene Round Bottom Tubes 70 mm cell strainers 30 mm celltrics strainers 50 ml Polypropylene conical tubes 15 ml Polypropylene conical tubes
11815275 16532 352063 352054 130-098-463 04-004-2326 352070 352097
Thermo Fisher Scientific Minisart BD Falcon BD Falcon Miltenyi Sysmex BD Falcon BD Falcon
080005 16532 130-098-463 130-098-463 352070 352097
Dominique Dutscher Minisart Miltenyi Miltenyi BD Falcon BD Falcon BD Biosciences
Human muscle cell isolation 30 ml Polystyrene Sterilin tube 0.22 mm filters 70 mm cell strainers 100 mm cell strainers 50 ml Polypropylene conical tubes 15 ml Polypropylene conical tubes FACS ARIA III
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3. 4.
5.
6. 7.
8. 9.
10. 11. 12. 13. 14.
Note: rapid dissection is an important parameter to obtain a good yield. Dissection must be performed on ice and should last less than 5 min, otherwise the number of viable cells strongly decreases. Place the muscles in Polypropylene FACS tubes and mince with scissors to obtain a pulp. Add the digestion medium and incubate the muscle pulp for 30 min in a gently shaking water bath at 37 °C. Every 10 min, vigorously shake the mixture by pipetting. Note: digestion duration was set up for normal regenerating Tibialis Anterior muscle after CTX injection. This time may be adjusted depending on the muscle to be digested, the type of injury or the genotype/phenotype of the mouse (for instance 45 min are required for a better digestion of fibrotic mdx muscle). Stop the digestion by adding Fetal Bovine Serum (FBS) (1 ml for 2 ml of digestion medium), homogenize and place the tube on ice. Keep the samples at 4 °C until the end of the procedure. Add 2 ml of serum-free DMEM/F12 and homogenize. Filtrate the digested muscle through a 70 mm cell strainer. Rinke the tube and the cell strainer with 2 ml of DMEM/ F12 serum-free. Centrifuge at 350g for 7 min at 4 °C. Discard the supernatant and lyse red cells with 0.5 ml of ACK Buffer. Vortex until the solution gets a pink/red color (3 to 5 s) and stop the lysis with 5 ml of 1X PBS containing 2% FBS. Note: Carefully monitor the time the cells are exposed to ACK lysis buffer to avoid prolonged incubation that would damage the cells of interest. Centrifuge at 350g for 7 min at 4 °C. Homogenize the pellet into 2 ml of 1X PBS containing 2% FBS. Centrifuge at 350g for 7 min at 4 °C. Add 1 ml 1X PBS containing 2% FBS. Count the cells and evaluate their viability (e.g. with Trypan blue dye).
2.2.2. Cell sorting 1. Distribute about 30,000 cells in polystyrene FACS tubes for several controls: Isotype control, Single staining control and FMO (Fluorescence Minus One) control, and distribute the rest of the cell suspension into the polystyrene FACS tube for sorting. Isotype control tube contains all isotype control antibodies relative to all fluorochromes. This tube is mandatory to confirm the specificity of primary antibody binding (see Table 2). Single staining control tube contains a single specific antibody (here 6 different tubes containing each a different antibody). These tubes are required to adjust the compensation on the flow cytometer, which eliminates false signal resulting from spectral overlap between fluorescent dyes. Fluorescence minus one (FMO) control tube contains all specific antibodies but one (here 6 different tubes are required) and are required to adjust the gates on the flow cytometer. 2. Centrifuge the tubes at 350g for 7 min at 4 °C. 3. Discard the supernatant and leave about 100 ll, then homogenize the cell pellet. 4. Add 2 ml of FcBlock for up to 1.106 cells and incubate for 30 min at 4 °C. 5. Add the antibodies (quantities are indicated in Table 3). 6. 20 min is the minimum incubation time, which may be up to 30–45 min. 7. Add 2 ml of PBS containing 2% FBS. 8. Centrifuge at 350g for 7 min at 4 °C.
Table 3 Quantity of Antibodies. Murine muscle cell isolation Antibodies Anti Anti Anti Anti Anti Anti Anti
a7 integrin-647 CD34-FITC CD45-PE CD31-eFluor450 Sca1-PerCP-Cy5.5 F4/80 APC-eFluor780 CD140a PE-Cy7
Quantity for 2 TA 2.5 mg 1.25 mg 0.1 mg 0.1 mg 0.1 mg 0.25 mg 0.6 mg
Human muscle cell isolation Antibodies
Quantity (mg)
Anti CD31-FITC Anti CD56-APC
0.5 mg 10 ml
9. Homogenize the pellet with 0.5 ml of 1X PBS containing 2% FBS, filter it through a 30 mm celltrics strainer just before sorting. Macrophages are sorted as CD45pos F4/80pos cells. CD45neg F4/80neg cell population includes the 3 other cells types: ECs are Sca1pos CD31pos CD34pos; SCs are Sca1neg CD31neg CD34pos a7 integrinpos and FAPs are Sca1pos CD31neg CD34pos CD140pos. Note: the yield of cells recovered is highly variable depending on the context. Basically, in normal non injured TA muscle, 50,000 to 70,000 ECs, 30,000 to 45,000 SCs, 300 to 1100 macrophages and 300 to 1500 FAPs are usually recovered from 1 mouse (2 TA muscles). 4 days after injury, 50,000 to 70,000 ECs, SCs, macrophages and FAPs are usually recovered. 2.3. Purification test To control the purity of the sorted populations, sorted cells may be re-analyzed with the flow cytometer, to evaluate the % of purity, or may be cytospined and labeled with primary antibodies against Pax7 for SCs, VE-cad for ECs, alpha-SMA for FAPs and CD11b for macrophages. 3. Human muscle cell isolation Fresh human biopsies are directly prepared for cell sorting for the isolation of ECs (as CD31pos CD56neg cells) and of SCs (as CD31neg CD56pos cells). Human samples were obtained after institutionally approved protocol and parents or legal representatives gave their written informed consent for the children’s participation to the study (protocol registered at the Ministère de la Recherche and Cochin Hospital Cell Bank, Paris, agreement n°DC-2009-944). All materials and reagents used are listed in Tables 1 and 2, respectively. 3.1. Tissue preparation 1. Prior dissection, prepare digestion buffer (collagenase B 1 mg/ ml – dispase II 2.4 U/ml) in ECGMV2 serum-free (10 ml/g of muscle). Heat the buffer at 37 °C and filtrate with 0.22 mm filter. 2. Fresh human biopsies (100–1000 mg) are finely minced, and digested for 30 to 45 min, depending on the size of the biopsy, in a water bath at 37 °C under continuous gentle shaking. Every 15 min, vigorously shake the mixture by pipetting. Note: the incubation time depends on the size and of the quality of the biopsy. Usually 30 min are sufficient. This time may be increased if the muscle is damaged (e.g. fibrotic) or if too few cells are recovered.
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Fig. 1. EC, SC, Macrophage and FAP isolation from mouse Tibialis anterior muscle. ECs, SCs, Macrophages (Mps) and FAPs were FACS-sorted from mouse regenerating skeletal muscle and analyzed. (A) Gating strategy: (A1) Total cell population. (A2) macrophages (Mps) were sorted as CD45posF4/80pos cells. (A3) Among CD45negF4/ 80neg cells gated in A2, FAPs, SCs and ECs were sorted using Sca1 and CD31 labelings. Sca1negCD31neg cells in A3 were further sorted as CD34loa7integrinpos SCs (A4), Sca1negCD31pos as CD34pos ECs (A5), and Sca1posCD31neg as CD140pos FAPs (A6). (B–E) Plots show the purity of the sorted populations. (B1) Purity of sorted Mps. (C1–C2) Purity of sorted SCs. (E1–E2) Purified of sorted FAPs. (F1–F4) Sorted cells were cytospined and immunostained with CD45 (F1), Pax7 (F2), CD31 (F3) or CD140a (F4) antibodies to assess the purity of macrophages, SCs, ECs and FAPs respectively (Cy3 = antibodies and Blue = Hoechst). Bar = 20 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. FMO control. Muscles and cells were processed as described in Fig. 1. On the left panel, all antibodies minus antiCD140a (FMO) were added. On the right panel all antibodies were added. Numbers refer to the indicated gates. The histograms in the bottom show no labeling for CD140a in the FMO tube.
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Fig. 3. EC and SC isolation from human muscle. Left panel: FSC/SSC plot of the cells isolated from human muscle. Middle panel: isotypic control shows no specific labeling. Right panel: gating showing the specific isolated cell populations: SCs (CD56posCD31neg) and ECs (CD56negCD31pos).
3. Stop the digestion by adding 10 ml of ECGMV2 containing 10% FBS and then filter through 100 mm then through 70 mm cell strainers to obtain a single cell suspension. 4. Centrifuge for 10 min at 400g at +4 °C. 5. Discard the supernatant and homogenize the pellet in PBS 1X containing 5% FBS (1–2 ml depending on the size of the biopsy). Count the cells and evaluate their viability (e.g. with Trypan blue dye). 3.2. Cell sorting 1. Distribute 80 ml of cell suspension in polystyrene FACS tubes for isotype controls and the rest of suspension into the polystyrene FACS tube for sorting. 2. Centrifuge for 10 min at 400g at +4 °C. 3. Add isotype antibodies in the control tubes and primary antibodies in the sorting tube (quantities are given in Table 3). 4. Incubate for 45 min at 4 °C protected from light. 5. Add 500 ll of PBS 1X containing 5% FBS. 6. Centrifuge for 10 min at 400g at +4 °C. 7. Discard the supernatant and homogenize the pellet with 300 ll of PBS 1X containing 5% FBS. 8. Cell sort the two cell populations. Note: the yield depends on the quality of the muscle but is still highly variable from one donor to the other. We recovered from 5000 to 25,000 SCs per g of muscle and 20,000 to 40,000 ECs per g of normal muscle. These yields changed in diseased muscles. 4. Results and discussion Our protocol allows to sort several cell types from regenerating mouse during skeletal muscle: ECs, SCs, macrophages and FAPs. The gating strategy allowed to sort macrophages as CD45posF4/80pos cells, ECs as CD45negF4/80negSca1posCD31posCD34pos, SCs as CD45negF4/80negSca1negCD31negCD34loa7-integrinpos and FAPs as CD45negF4/80negSca1posCD31negCD140pos (PDGFRa) (Fig. 1A). To sort pure populations, stringent gates were drawn. The purity of the sorted cell populations was tested by FACs analysis. Plots in Fig. 1B1–E2 show the purity of the sorted cell populations. Regarding sorted macrophage population cells presenting a low F4/80 staining were observed, since the level of F4/80 expression may vary according to the maturation of macrophages within the regenerating skeletal muscle [4]. Moreover, the purity of the sorted cells was confirmed by immunocytochemistry performed on cytospin preparations of the sorted cells: CD31 and VE-cad labeling for ECs, Pax7 labeling for MPCs, CD45 labeling for macrophages and CD140a labeling for FAPs (Fig. 1F). FMO controls allowed to assess the specificity of the labeling. An example is given for CD140a (Fig. 2). Sorted cells can be further used to perform several kinds of analysis. They may be used for direct molecular analysis by RT-
qPCR, microarray, RNA sequencing or single cell analysis. For molecular analysis, freshly isolated cells are immediately centrifuged and homogenized in trio reagent to extract RNAs. Functional experiments can be also performed using the freshly sorted cells using cell culture or cell co-cultures. These are usually performed in 96 well plates given the low amounts of cells that are recovered. Nevertheless, several behaviors of the cells can be monitored such as cell proliferation, cell differentiation, secretion of cytokines/chemokines (ELISA), etc. Our protocol from human muscle sample allows the isolation of ECs and SCs (Fig. 3) from relatively small biopsy samples, that can be further used for molecular analysis (microarray, RNAseq, etc.) [15]. Cell sorting has becoming of a great interest for the study of various cell types extracted from tissues. One limitation of cell sorting resides into what extent the digestion and sorting procedure affects the status of the cells. However, comparison of cells issued from tissues having different status (e.g. during repair process, or normal versus disease, or WT versus KO) will surely give insights on the biological processes involving these cell types. Another issue is the number of cells that can be recovered from cell sorting to perform further experiments, and that may require numerous mice (cells from several mice can be pooled), which may run counter to ethic rules. Nevertheless, besides classic culture and cocultures experiments using cell lines or conventional primary cultures, the use of cell sorted cell populations is essential for validating biological processes and their underlying cell interactions and molecular pathways. 5. Conclusions Regarding the complexity of the cellular interactions that occur during tissue repair and disease, FACS sorting of pure populations of the various cell types involved in these processes is of high interest. Evaluation of cells at the molecular and functional level from a variety of sources (normal, pathological, KO, KI) can be achieved in a high throughput manner using this technology. Declaration of interest The authors declare no competing financial interests Author contributions Conceptualization: BC, CL, MWG; Methodology: BC, CL, MWG, CG; Validation: BC, CL, MWG, RM; Formal Analysis: BC, CL, MWG, RM; Investigation: CL, MWG, CG; Resources: BC, RM; Data Curation: BC, CL, MWG; Writing, Original Draft: BC, CL, MWG; Writing, Review & Editing: BC, CL, MWG, RM; Visualization: BC, CL; Supervision: BC, RM; Project Administration: BC; Funding Acquisition: BC.
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Acknowledgments/Fundings This work was funded by Inserm, CNRS, Université Paris Descartes and Université Claude Bernard Lyon 1 (France), by EU FP7 Endostem (#241440) and by Association Française contre les Myopathies (grant #18003). CL was supported by Dim Stem Pole from Région Ile-de-France and Association Française contre les Myopathies. CG was supported by a Poste d’Accueil APHP/CNRS. We thank AniRA-Cytometry facility form SFR Biosciences, Université Claude Bernard Lyon 1 and Cybio facility from Institut Cochin. References [1] H. Yin, F. Price, M.A. Rudnicki, Satellite cells and the muscle stem cell niche, Physiol. Rev. 93 (1) (2013) 23–67. [2] R. Sambasivan, R. Yao, A. Kissenpfennig, L. Van Wittenberghe, A. Paldi, B. Gayraud-Morel, H. Guenou, B. Malissen, S. Tajbakhsh, A. Galy, Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration, Development 138 (17) (2011) 3647–3656. [3] C. Lepper, T.A. Partridge, C.-M. Fan, An absolute requirement for Pax7-positive satellite cells in acute injury-induced skeletal muscle regeneration, Development 138 (17) (2011) 3639–3646. [4] L. Arnold, A. Henry, F. Poron, Y. Baba-Amer, N. van Rooijen, A. Plonquet, R.K. Gherardi, B. Chazaud, Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis, J. Exp. Med. 204 (5) (2007) 1057–1069. [5] M. Saclier, H. Yacoub-Youssef, A.L. Mackey, L. Arnold, H. Ardjoune, M. Magnan, F. Sailhan, J. Chelly, G.K. Pavlath, R. Mounier, M. Kjaer, B. Chazaud, Differentially activated macrophages orchestrate myogenic precursor cell fate during human skeletal muscle regeneration, Stem Cells 31 (2) (2013) 384– 396. [6] R. Mounier, F. Chretien, B. Chazaud, Blood vessels and the satellite cell niche, Curr. Top. Dev. Biol. 96 (2011) 121–138.
[7] C. Christov, F. Chretien, R. Abou-Khalil, G. Bassez, G. Vallet, F.J. Authier, Y. Bassaglia, V. Shinin, S. Tajbakhsh, B. Chazaud, R.K. Gherardi, Muscle satellite cells and endothelial cells: close neighbors and privileged partners, Mol. Biol. Cell 18 (4) (2007) 1397–1409. [8] R. Abou-Khalil, F. Le Grand, G. Pallafacchina, S. Valable, F.-J. Authier, M.A. Rudnicki, R.K. Gherardi, S. Germain, F. Chretien, A. Sotiropoulos, P. Lafuste, D. Montarras, B. Chazaud, Autocrine and paracrine angiopoietin 1/Tie-2 signaling promotes muscle satellite cell self-renewal, Cell Stem Cell 5 (3) (2009) 298– 309. [9] M.M. Murphy, J.A. Lawson, S.J. Mathew, D.A. Hutcheson, G. Kardon, Satellite cells, connective tissue fibroblasts and their interactions are crucial for muscle regeneration, Development 138 (17) (2011) 3625–3637. [10] A.W.B. Joe, L. Yi, A. Natarajan, F. Le Grand, L. So, J. Wang, M.A. Rudnicki, F.M.V. Rossi, Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis, Nat. Cell Biol. 12 (2) (2010) 153–163. [11] A. Uezumi, S. Fukada, N. Yamamoto, S. Takeda, K. Tsuchida, Mesenchymal progenitors distinct from satellite cells contribute to ectopic fat cell formation in skeletal muscle, Nat. Cell Biol. 12 (2) (2010) 143–152. [12] D. Hardy, A. Besnard, M. Latil, G. Jouvion, D. Briand, C. Thépenier, Q. Pascal, A. Guguin, B. Gayraud-Morel, J.-M. Cavaillon, S. Tajbakhsh, P. Rocheteau, F. Chrétien, Comparative study of injury models for studying muscle regeneration in mice, PLoS One 11 (1) (2016) e0147198. [13] T. Varga, R. Mounier, A. Horvath, S. Cuvellier, F. Dumont, S. Poliska, H. Ardjoune, G. Juban, L. Nagy, B. Chazaud, Highly dynamic transcriptional signature of distinct macrophage subsets during sterile inflammation, resolution, and tissue repair, J. Immunol. 196 (11) (Jun. 2016) 4771–4782. [14] R. Mounier, M. Théret, L. Arnold, S. Cuvellier, L. Bultot, O. Göransson, N. Sanz, A. Ferry, K. Sakamoto, M. Foretz, B. Viollet, B. Chazaud, AMPKa1 regulates macrophage skewing at the time of resolution of inflammation during skeletal muscle regeneration, Cell Metab. 18 (2) (2013) 251–264. [15] C. Gitiaux, C. Latroche, M. Weiss-Gayet, M.P. Rodero, D. Duffy, B. Bader Meunier, C. Glorion, P. Nusbaum, C. Bodemer, G. Mouchiroud, J. Chelly, S. Germain, I. Desguerre, B. Chazaud, Myogenic progenitor cells exhibit IFN type I-driven pro-angiogenic properties and molecular signature during juvenile dermatomyositis, Arthritis Rheumatol. (2017), https://doi.org/10.1002/ art.40328.
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Methods journal homepage: www.elsevier.com/locate/ymeth
Cell sorting of various cell types from mouse and human skeletal muscle Claire Latroche a, Michèle Weiss-Gayet b, Cyril Gitiaux a, Bénédicte Chazaud b,⇑ a b
Institut Cochin, INSERM U1016, CNRS UMR8104, Université Paris Descartes, PRES Sorbonne-Paris-Cité, Paris, France Institut NeuroMyoGène, Univ Lyon, Université Claude Bernard Lyon 1, INSERM U1217, CNRS UMR5310, Lyon, France
a r t i c l e
i n f o
Article history: Received 30 June 2017 Received in revised form 11 December 2017 Accepted 15 December 2017 Available online xxxx Keywords: Skeletal muscle regeneration Cell sorting Myogenic cells Fibroblasts Macrophages Endothelial cells
a b s t r a c t Muscle stem cells or satellite cells are required for skeletal muscle regeneration. It has been shown that the satellite cell microenvironment, including neighboring cells such as endothelial cells, macrophages or fibroblasts are essential for complete and efficient regeneration. A deficient behavior of these cells compromises regeneration. Therefore, there is a strong interest in understanding the cellular and molecular interactions at work between these cell types during muscle regeneration. Fluorescence-activated cell sorting allows to isolate these four cell types at different time points of regeneration, for further high throughput or behavioral experiments. We present here a method for the concomitant isolation of 4 cell types present in the regenerating skeletal muscle: muscle stem cells, endothelial cells, fibro-adipogenic precursor cells and macrophages. Ó 2017 Elsevier Inc. All rights reserved.
1. Introduction Normal adult skeletal muscle regenerates ad integrum after injury. In experimental injury models in mouse, muscle tissue undergoes necrosis followed by rapid infiltration of immune cells. Few days later (depending on the type and extent of the injury), new myofibers are formed. After about one month, regenerating muscle is similar to uninjured muscle except that the myofibers exhibit central nuclei. Satellite cells (SCs) are the principal contributors for the regeneration of skeletal muscle. Upon myofiber damage, quiescent SCs become activated, divide, and give rise to a population of transient amplifying myogenic precursor cells (skeletal myoblasts). Later on, most of these myogenic precursors exit the cell cycle, lose Pax7 expression, differentiate and fuse with the host fibers or between them to form new myofibers. A subset of myogenic precursor does not differentiate and self-renews to replenish the pool of SCs [1]. While SCs are indispensable for skeletal muscle regeneration [2,3], other cell types, such as fibroblasts, endothelial cells and immune cells, among which macrophages, have been shown to play important roles in this process. Indeed, these cells establish specific interactions with SCs to ensure efficient muscle regeneration. However, the cell dynamics are highly complex and occur with specific temporal and spatial kinetics. ⇑ Corresponding author at: Institut NeuroMyoGène, Université Claude Bernard Lyon 1, 8 Avenue Rockfeller, 69003 Lyon, France. E-mail address: [email protected] (B. Chazaud).
Immune cells invade the muscle early after injury, first as neutrophils, then as macrophages. Macrophages serve as key effectors in the muscle stem cell niche to guide SCs through the regeneration process through direct interactions with myogenic cells. They first invade the muscle as inflammatory macrophages (coming from inflammatory circulating monocytes) and specifically stimulate myogenic precursor proliferation and trigger Fibro-Adipogenic Precursor (FAP) apoptosis (see below). Then, upon the phagocytosis of necrotic muscle debris, they switch their inflammatory status into anti-inflammatory cells, that stimulate myogenic differentiation and promote myofiber growth [4–6]. Skeletal muscle is laced with a dense microvasculature and most quiescent SCs are located close to capillaries. Vascular cells also coordinate both the acute SC response and the late stages of muscle regeneration when the tissue returns to homeostasis. Endothelial cells (ECs) predominantly exhibit pro-myogenic effects on SCs, through a strong stimulating effect on their differentiation, while peri-endothelial cells, such as smooth muscle cells and fibrogenic cell types, are crucial for the re-entry into quiescence on completion of regeneration. Inversely, SCs and myogenic precursors have been shown to exhibit pro-angiogenic activity [6–8]. Finally, fibroblasts and FAPs are the main source of matrix proteins during muscle regeneration and are required for muscle regeneration [9]. Upon muscle injury, FAPs are activated, rapidly expand and control matrix remodeling, while providing an environment favoring myogenic differentiation [10,11]. While these observations show the existence of specific interactions between SCs and the neighboring cells during muscle
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regeneration, the molecular mechanisms involved in this complex network of communication are still poorly known. In this study, we describe a protocol for isolating pure populations of SCs, macrophages, ECs and FAPs from regenerating mouse skeletal muscle and for isolating SCs and ECS from human muscle samples.
2. Isolation of murine muscle cells
2.2.1. Cell preparation 1. Prior dissection, prepare the digestion medium (collagenase B 10 mg/ml – dispase II 2,4U/ml) in DMEM/F12 serum-free (1 ml/muscle). Heat the medium at 37 °C and filtrate through a 0.22 mm filter. 2. Dissect Tibialis Anterior of both hind limbs. In a Petri dish on ice, roughly discard visible fat, tendons and fascia.
2.1. Induction of muscle regeneration Muscle injury was performed by the injection of cardiotoxin (CTX), a kinase C inhibitor extracted from Anja nigricollis snake venom, that induces depolarization and contraction of muscle cells and destroys the cell membrane structure [12]. CTX injection provides an homogeneous damage in the whole muscle, and the following sequential steps of the regeneration process are well described [13]. Intramuscular injection of CTX (12 mM, 50 ml, Latoxan, France) was performed in the Tibialis Anterior muscle of adult male mice [14]. Adult 6–8 week-old male C57Bl/6 mice were bred and used in compliance with French and European regulations. Principal investigator is licensed for these experiments and the protocols were approved by local Animal Care and Use Committee and the French Ministry of Agriculture. Usually, the time points studied are days 1, 2, 4 and 8 after injury. At day 1 after injury, myofibers are necrotic, while immune cells (neutrophils and circulating monocytes) enter into the damaged muscle. At day 2, macrophages phagocyte the dead myofibers and FAPs are highly expanding while SCs are also in the amplification phase. At day 4 after injury, the regeneration process is visible with the appearance of the new regenerating myofibers indicative of myogenic differentiation. Macrophages are still numerous in supporting this process, while FAPs are remodeling the matrix and angiogenesis takes place. At day 8, muscle has almost recovered its original appearance, with nanofibers characterized by the central location of their nuclei, microvasculature maturing and the almost disappearance of macrophages and FAPs.
2.2. Flow cytometry
Table 2 Reagents. Reagent
Cat. No
Manufacturer
Murine muscle cell isolation DMEM/F12 Collagenase B 10 mg/ml Dispase II 2.4 U/ml Fetal Bovine Serum ACK Buffer 1X PBS Fc block Anti a7 integrin-647 Anti CD34-FITC Anti CD45-PE Anti CD31-eFluor450 Anti Sca1-PerCP-Cy5.5 Anti F4/80 APC-eFluor780 Anti CD140a PE-Cy7 Rat IgG2a Isotype control FITC Rat IgG2b Isotype control PE Rat IgG2b Isotype control eFluor 450 Rat IgG2a Isotype control PerCP-Cy5-5 Rat IgG2a Isotype control APC-eFluor 780 Rat IgG2a Isotype control PE-Cy7
31331-028 11 088 831 001 04 942 078 001 10270-106 10-548E 14190-094 130-059-901 AB0000538 11-0341 12-0451 48-0311 45-5981 47-4801 25-1401 11-4321 12-4031 48-4031 45-4321 47-4321 25-4321
Gibco Roche Roche Gibco Lonza Gibco Miltenyi AB lab eBioscience eBioscience eBioscience eBioscience eBioscience eBioscience eBioscience eBioscience eBioscience eBioscience eBioscience eBioscience
Human muscle cell isolation ECGMV2 Collagenase B 1 mg/ml Dispase II 2.4 U/ml Fetal Bovine Serum 1X PBS Anti CD31-FITC Anti CD56-APC
C-22022 11 088 831 001 04 942 078 001 10270-106 14190-094 11-0319 555518
Mouse IgG1 Isotype Control FITC Mouse IgG1 Isotype Control APC
11-4714 555751
Promocell Roche Roche Gibco Gibco eBioscience BD Pharmingen eBioscience BD Pharmingen
All materials and reagents used are listed in Tables 1 and 2 respectively.
Table 1 Material. Material
Cat. No
Manufacturer
Murine muscle cell isolation Material for mouse dissection (thin forceps, rasor blade or thin scissors, thin sharp scissors) Petri dishes 0.22 mm filters Polypropylene Round Bottom Tube Polystyrene Round Bottom Tubes 70 mm cell strainers 30 mm celltrics strainers 50 ml Polypropylene conical tubes 15 ml Polypropylene conical tubes
11815275 16532 352063 352054 130-098-463 04-004-2326 352070 352097
Thermo Fisher Scientific Minisart BD Falcon BD Falcon Miltenyi Sysmex BD Falcon BD Falcon
080005 16532 130-098-463 130-098-463 352070 352097
Dominique Dutscher Minisart Miltenyi Miltenyi BD Falcon BD Falcon BD Biosciences
Human muscle cell isolation 30 ml Polystyrene Sterilin tube 0.22 mm filters 70 mm cell strainers 100 mm cell strainers 50 ml Polypropylene conical tubes 15 ml Polypropylene conical tubes FACS ARIA III
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3. 4.
5.
6. 7.
8. 9.
10. 11. 12. 13. 14.
Note: rapid dissection is an important parameter to obtain a good yield. Dissection must be performed on ice and should last less than 5 min, otherwise the number of viable cells strongly decreases. Place the muscles in Polypropylene FACS tubes and mince with scissors to obtain a pulp. Add the digestion medium and incubate the muscle pulp for 30 min in a gently shaking water bath at 37 °C. Every 10 min, vigorously shake the mixture by pipetting. Note: digestion duration was set up for normal regenerating Tibialis Anterior muscle after CTX injection. This time may be adjusted depending on the muscle to be digested, the type of injury or the genotype/phenotype of the mouse (for instance 45 min are required for a better digestion of fibrotic mdx muscle). Stop the digestion by adding Fetal Bovine Serum (FBS) (1 ml for 2 ml of digestion medium), homogenize and place the tube on ice. Keep the samples at 4 °C until the end of the procedure. Add 2 ml of serum-free DMEM/F12 and homogenize. Filtrate the digested muscle through a 70 mm cell strainer. Rinke the tube and the cell strainer with 2 ml of DMEM/ F12 serum-free. Centrifuge at 350g for 7 min at 4 °C. Discard the supernatant and lyse red cells with 0.5 ml of ACK Buffer. Vortex until the solution gets a pink/red color (3 to 5 s) and stop the lysis with 5 ml of 1X PBS containing 2% FBS. Note: Carefully monitor the time the cells are exposed to ACK lysis buffer to avoid prolonged incubation that would damage the cells of interest. Centrifuge at 350g for 7 min at 4 °C. Homogenize the pellet into 2 ml of 1X PBS containing 2% FBS. Centrifuge at 350g for 7 min at 4 °C. Add 1 ml 1X PBS containing 2% FBS. Count the cells and evaluate their viability (e.g. with Trypan blue dye).
2.2.2. Cell sorting 1. Distribute about 30,000 cells in polystyrene FACS tubes for several controls: Isotype control, Single staining control and FMO (Fluorescence Minus One) control, and distribute the rest of the cell suspension into the polystyrene FACS tube for sorting. Isotype control tube contains all isotype control antibodies relative to all fluorochromes. This tube is mandatory to confirm the specificity of primary antibody binding (see Table 2). Single staining control tube contains a single specific antibody (here 6 different tubes containing each a different antibody). These tubes are required to adjust the compensation on the flow cytometer, which eliminates false signal resulting from spectral overlap between fluorescent dyes. Fluorescence minus one (FMO) control tube contains all specific antibodies but one (here 6 different tubes are required) and are required to adjust the gates on the flow cytometer. 2. Centrifuge the tubes at 350g for 7 min at 4 °C. 3. Discard the supernatant and leave about 100 ll, then homogenize the cell pellet. 4. Add 2 ml of FcBlock for up to 1.106 cells and incubate for 30 min at 4 °C. 5. Add the antibodies (quantities are indicated in Table 3). 6. 20 min is the minimum incubation time, which may be up to 30–45 min. 7. Add 2 ml of PBS containing 2% FBS. 8. Centrifuge at 350g for 7 min at 4 °C.
Table 3 Quantity of Antibodies. Murine muscle cell isolation Antibodies Anti Anti Anti Anti Anti Anti Anti
a7 integrin-647 CD34-FITC CD45-PE CD31-eFluor450 Sca1-PerCP-Cy5.5 F4/80 APC-eFluor780 CD140a PE-Cy7
Quantity for 2 TA 2.5 mg 1.25 mg 0.1 mg 0.1 mg 0.1 mg 0.25 mg 0.6 mg
Human muscle cell isolation Antibodies
Quantity (mg)
Anti CD31-FITC Anti CD56-APC
0.5 mg 10 ml
9. Homogenize the pellet with 0.5 ml of 1X PBS containing 2% FBS, filter it through a 30 mm celltrics strainer just before sorting. Macrophages are sorted as CD45pos F4/80pos cells. CD45neg F4/80neg cell population includes the 3 other cells types: ECs are Sca1pos CD31pos CD34pos; SCs are Sca1neg CD31neg CD34pos a7 integrinpos and FAPs are Sca1pos CD31neg CD34pos CD140pos. Note: the yield of cells recovered is highly variable depending on the context. Basically, in normal non injured TA muscle, 50,000 to 70,000 ECs, 30,000 to 45,000 SCs, 300 to 1100 macrophages and 300 to 1500 FAPs are usually recovered from 1 mouse (2 TA muscles). 4 days after injury, 50,000 to 70,000 ECs, SCs, macrophages and FAPs are usually recovered. 2.3. Purification test To control the purity of the sorted populations, sorted cells may be re-analyzed with the flow cytometer, to evaluate the % of purity, or may be cytospined and labeled with primary antibodies against Pax7 for SCs, VE-cad for ECs, alpha-SMA for FAPs and CD11b for macrophages. 3. Human muscle cell isolation Fresh human biopsies are directly prepared for cell sorting for the isolation of ECs (as CD31pos CD56neg cells) and of SCs (as CD31neg CD56pos cells). Human samples were obtained after institutionally approved protocol and parents or legal representatives gave their written informed consent for the children’s participation to the study (protocol registered at the Ministère de la Recherche and Cochin Hospital Cell Bank, Paris, agreement n°DC-2009-944). All materials and reagents used are listed in Tables 1 and 2, respectively. 3.1. Tissue preparation 1. Prior dissection, prepare digestion buffer (collagenase B 1 mg/ ml – dispase II 2.4 U/ml) in ECGMV2 serum-free (10 ml/g of muscle). Heat the buffer at 37 °C and filtrate with 0.22 mm filter. 2. Fresh human biopsies (100–1000 mg) are finely minced, and digested for 30 to 45 min, depending on the size of the biopsy, in a water bath at 37 °C under continuous gentle shaking. Every 15 min, vigorously shake the mixture by pipetting. Note: the incubation time depends on the size and of the quality of the biopsy. Usually 30 min are sufficient. This time may be increased if the muscle is damaged (e.g. fibrotic) or if too few cells are recovered.
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Fig. 1. EC, SC, Macrophage and FAP isolation from mouse Tibialis anterior muscle. ECs, SCs, Macrophages (Mps) and FAPs were FACS-sorted from mouse regenerating skeletal muscle and analyzed. (A) Gating strategy: (A1) Total cell population. (A2) macrophages (Mps) were sorted as CD45posF4/80pos cells. (A3) Among CD45negF4/ 80neg cells gated in A2, FAPs, SCs and ECs were sorted using Sca1 and CD31 labelings. Sca1negCD31neg cells in A3 were further sorted as CD34loa7integrinpos SCs (A4), Sca1negCD31pos as CD34pos ECs (A5), and Sca1posCD31neg as CD140pos FAPs (A6). (B–E) Plots show the purity of the sorted populations. (B1) Purity of sorted Mps. (C1–C2) Purity of sorted SCs. (E1–E2) Purified of sorted FAPs. (F1–F4) Sorted cells were cytospined and immunostained with CD45 (F1), Pax7 (F2), CD31 (F3) or CD140a (F4) antibodies to assess the purity of macrophages, SCs, ECs and FAPs respectively (Cy3 = antibodies and Blue = Hoechst). Bar = 20 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. FMO control. Muscles and cells were processed as described in Fig. 1. On the left panel, all antibodies minus antiCD140a (FMO) were added. On the right panel all antibodies were added. Numbers refer to the indicated gates. The histograms in the bottom show no labeling for CD140a in the FMO tube.
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Fig. 3. EC and SC isolation from human muscle. Left panel: FSC/SSC plot of the cells isolated from human muscle. Middle panel: isotypic control shows no specific labeling. Right panel: gating showing the specific isolated cell populations: SCs (CD56posCD31neg) and ECs (CD56negCD31pos).
3. Stop the digestion by adding 10 ml of ECGMV2 containing 10% FBS and then filter through 100 mm then through 70 mm cell strainers to obtain a single cell suspension. 4. Centrifuge for 10 min at 400g at +4 °C. 5. Discard the supernatant and homogenize the pellet in PBS 1X containing 5% FBS (1–2 ml depending on the size of the biopsy). Count the cells and evaluate their viability (e.g. with Trypan blue dye). 3.2. Cell sorting 1. Distribute 80 ml of cell suspension in polystyrene FACS tubes for isotype controls and the rest of suspension into the polystyrene FACS tube for sorting. 2. Centrifuge for 10 min at 400g at +4 °C. 3. Add isotype antibodies in the control tubes and primary antibodies in the sorting tube (quantities are given in Table 3). 4. Incubate for 45 min at 4 °C protected from light. 5. Add 500 ll of PBS 1X containing 5% FBS. 6. Centrifuge for 10 min at 400g at +4 °C. 7. Discard the supernatant and homogenize the pellet with 300 ll of PBS 1X containing 5% FBS. 8. Cell sort the two cell populations. Note: the yield depends on the quality of the muscle but is still highly variable from one donor to the other. We recovered from 5000 to 25,000 SCs per g of muscle and 20,000 to 40,000 ECs per g of normal muscle. These yields changed in diseased muscles. 4. Results and discussion Our protocol allows to sort several cell types from regenerating mouse during skeletal muscle: ECs, SCs, macrophages and FAPs. The gating strategy allowed to sort macrophages as CD45posF4/80pos cells, ECs as CD45negF4/80negSca1posCD31posCD34pos, SCs as CD45negF4/80negSca1negCD31negCD34loa7-integrinpos and FAPs as CD45negF4/80negSca1posCD31negCD140pos (PDGFRa) (Fig. 1A). To sort pure populations, stringent gates were drawn. The purity of the sorted cell populations was tested by FACs analysis. Plots in Fig. 1B1–E2 show the purity of the sorted cell populations. Regarding sorted macrophage population cells presenting a low F4/80 staining were observed, since the level of F4/80 expression may vary according to the maturation of macrophages within the regenerating skeletal muscle [4]. Moreover, the purity of the sorted cells was confirmed by immunocytochemistry performed on cytospin preparations of the sorted cells: CD31 and VE-cad labeling for ECs, Pax7 labeling for MPCs, CD45 labeling for macrophages and CD140a labeling for FAPs (Fig. 1F). FMO controls allowed to assess the specificity of the labeling. An example is given for CD140a (Fig. 2). Sorted cells can be further used to perform several kinds of analysis. They may be used for direct molecular analysis by RT-
qPCR, microarray, RNA sequencing or single cell analysis. For molecular analysis, freshly isolated cells are immediately centrifuged and homogenized in trio reagent to extract RNAs. Functional experiments can be also performed using the freshly sorted cells using cell culture or cell co-cultures. These are usually performed in 96 well plates given the low amounts of cells that are recovered. Nevertheless, several behaviors of the cells can be monitored such as cell proliferation, cell differentiation, secretion of cytokines/chemokines (ELISA), etc. Our protocol from human muscle sample allows the isolation of ECs and SCs (Fig. 3) from relatively small biopsy samples, that can be further used for molecular analysis (microarray, RNAseq, etc.) [15]. Cell sorting has becoming of a great interest for the study of various cell types extracted from tissues. One limitation of cell sorting resides into what extent the digestion and sorting procedure affects the status of the cells. However, comparison of cells issued from tissues having different status (e.g. during repair process, or normal versus disease, or WT versus KO) will surely give insights on the biological processes involving these cell types. Another issue is the number of cells that can be recovered from cell sorting to perform further experiments, and that may require numerous mice (cells from several mice can be pooled), which may run counter to ethic rules. Nevertheless, besides classic culture and cocultures experiments using cell lines or conventional primary cultures, the use of cell sorted cell populations is essential for validating biological processes and their underlying cell interactions and molecular pathways. 5. Conclusions Regarding the complexity of the cellular interactions that occur during tissue repair and disease, FACS sorting of pure populations of the various cell types involved in these processes is of high interest. Evaluation of cells at the molecular and functional level from a variety of sources (normal, pathological, KO, KI) can be achieved in a high throughput manner using this technology. Declaration of interest The authors declare no competing financial interests Author contributions Conceptualization: BC, CL, MWG; Methodology: BC, CL, MWG, CG; Validation: BC, CL, MWG, RM; Formal Analysis: BC, CL, MWG, RM; Investigation: CL, MWG, CG; Resources: BC, RM; Data Curation: BC, CL, MWG; Writing, Original Draft: BC, CL, MWG; Writing, Review & Editing: BC, CL, MWG, RM; Visualization: BC, CL; Supervision: BC, RM; Project Administration: BC; Funding Acquisition: BC.
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Acknowledgments/Fundings This work was funded by Inserm, CNRS, Université Paris Descartes and Université Claude Bernard Lyon 1 (France), by EU FP7 Endostem (#241440) and by Association Française contre les Myopathies (grant #18003). CL was supported by Dim Stem Pole from Région Ile-de-France and Association Française contre les Myopathies. CG was supported by a Poste d’Accueil APHP/CNRS. We thank AniRA-Cytometry facility form SFR Biosciences, Université Claude Bernard Lyon 1 and Cybio facility from Institut Cochin. References [1] H. Yin, F. Price, M.A. Rudnicki, Satellite cells and the muscle stem cell niche, Physiol. Rev. 93 (1) (2013) 23–67. [2] R. Sambasivan, R. Yao, A. Kissenpfennig, L. Van Wittenberghe, A. Paldi, B. Gayraud-Morel, H. Guenou, B. Malissen, S. Tajbakhsh, A. Galy, Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration, Development 138 (17) (2011) 3647–3656. [3] C. Lepper, T.A. Partridge, C.-M. Fan, An absolute requirement for Pax7-positive satellite cells in acute injury-induced skeletal muscle regeneration, Development 138 (17) (2011) 3639–3646. [4] L. Arnold, A. Henry, F. Poron, Y. Baba-Amer, N. van Rooijen, A. Plonquet, R.K. Gherardi, B. Chazaud, Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis, J. Exp. Med. 204 (5) (2007) 1057–1069. [5] M. Saclier, H. Yacoub-Youssef, A.L. Mackey, L. Arnold, H. Ardjoune, M. Magnan, F. Sailhan, J. Chelly, G.K. Pavlath, R. Mounier, M. Kjaer, B. Chazaud, Differentially activated macrophages orchestrate myogenic precursor cell fate during human skeletal muscle regeneration, Stem Cells 31 (2) (2013) 384– 396. [6] R. Mounier, F. Chretien, B. Chazaud, Blood vessels and the satellite cell niche, Curr. Top. Dev. Biol. 96 (2011) 121–138.
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