Mouse Satellite Cell Isolation and Transplantation
小鼠卫星细胞的分离和移植   

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Stem Cells
Jul 2017

 

Abstract

Satellite cell (SC) transplantation represents a powerful strategy to investigate SC biology during muscle regeneration. We described here a protocol for SC isolation from green fluorescent protein (GFP)-expressing mice and their transplantation into murine muscles. This procedure was originally used to assess the effects of the hormone unacylated ghrelin on muscle regeneration, in particular evaluating how the increase of unacylated ghrelin in the recipient muscle affected the engraftment of donor SCs (Reano et al., 2017).

Keywords: Satellite cells (卫星细胞), Muscle stem cells (肌肉干细胞), Transplant (移植), Engraftment (植入), Skeletal muscle (骨骼肌), Regeneration (再生), Muscle excision (肌肉切除), Cardiotoxin (心脏毒素)

Background

Skeletal muscle, which is composed of differentiated myofibers, can regenerate upon injury. Muscle regeneration relies on a population of quiescent resident stem cells called satellite cells (SCs) that reside beneath the basal lamina of the muscle fiber (Mauro, 1961). Upon injury, SCs undergo activation, extensive proliferation, differentiation, and fusion, eventually repairing or replacing the damaged myofibers (Collins et al., 2005).

Transplantation of SCs was considered for many years a potential therapy for Duchenne Muscular Dystrophy (DMD), since the engrafted myoblast can fuse with host myoblasts, suggesting the possibility of a functional repair in defective fibers (Partridge et al., 1978). Unfortunately, in human clinical trials in DMD patients, this strategy failed to restore dystrophin in injected muscles, and no functional improvements have been observed (Partridge, 2002). Nevertheless, SCs transplantation still represents a robust strategy to investigate SC biology, mainly to study both cell-autonomous and non-cell-autonomous mechanisms of muscle regeneration. In this protocol, we describe a method to transplant cells isolated from GFP-expressing donor mice that allow an easy tracking and measurement of engrafted cells in the recipient muscles. We used a simple and relatively inexpensive cell isolation method; however, cells to be transplanted can be isolated by different techniques, such as Fluorescence Activated Cell Sorting (FACS) or magnetic beads.

Materials and Reagents

  1. 0.2 µm microfilters (VWR, catalog number: 28145-475 )
  2. Disposable sterile plastics:
    1. 15 ml conical tubes
    2. 100 mm Petri dishes
    3. 10 ml conical tubes
    4. 5 ml serological pipettes
    5. 10 ml serological pipettes
    6. 1.5 ml microcentrifuge tubes
    7. 20 to 1,000 µl pipette tips
    8. 10 ml plastic syringes (Terumo Medical, catalog number: SS-10L )
  3. Surgical blades (Moretti S.p.a.–Chimo)
  4. 40 µm cell-strainer (Corning, catalog number: 431750 )
  5. Adhesion microscope slides Superfrost Ultraplus (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: J3800AMNZ )
  6. 22 x 22 mm slide cover glasses (Bio-Optica, catalog number: 09-02222 )
  7. Feather microtome C35 blades (PFM Medical, catalog number: 207500003 )
  8. Blotting paper for general purpose
  9. 10 week-old C57BL/6-Tg(CAG-EGFP)131Osb/LeySopJ mice (GFP mice), as donor mice (THE JACKSON LABORATORY, catalog number: 006567 )
  10. 4 month-old C57BL/6J, as recipient mice (THE JACKSON LABORATORY, catalog number: 000664 )
  11. Isofluorane (Isofluorane-vet, Merial Italia, catalog number: 00018152 )
  12. Ethanol (Honeywell International, catalog number: 458600 )
  13. Liquid nitrogen
  14. Cryostat embedding medium, Killik (Bio-Optica, catalog number: 05-9801 )
  15. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153 )
  16. Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
  17. Rabbit anti-green fluorescent protein (GFP) antibody (Thermo Fisher Scientific, Invitrogen, catalog number: A-11122 )
  18. Donkey anti-rabbit IgG (H+L) Alexa Fluor 488 conjugate (Thermo Fisher Scientific, Invitrogen, catalog number: A-21206 )
  19. 4’,6-Diamidino-2-phenylindole, dihydrochloride (DAPI) (Thermo Fisher Scientific, InvitrogenTM, catalog number: D1306 )
  20. Slow-fade, glycerol-based mounting medium (Thermo Fisher Scientific, InvitrogenTM, catalog number: S36936 )
  21. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
  22. Sucrose (Sigma-Aldrich, catalog number: S0389 )
  23. Cardiotoxin (CTX) from Naja pallida (Latoxan, catalog number: L8102 )
  24. 0.9% NaCl (Sigma-Aldrich, catalog number: S8776 )
  25. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
  26. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
  27. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: 255793 )
  28. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
  29. Pronase from Streptomyces griseus (Sigma-Aldrich, Roche Diagnostics, catalog number: 10165921001 )
  30. DMEM–Dulbecco’s Modified Eagle Medium, high glucose (Thermo Fisher Scientific, GibcoTM, catalog number: 11965092 ) supplemented (for all procedures) with antibiotic-antimycotic solution (Sigma-Aldrich, catalog number: A5955 )
  31. Donor horse serum (HS) (Thermo Fisher Scientific, GibcoTM, catalog number: 16050130 )
  32. Fetal bovine serum, FBS (Thermo Fisher Scientific, GibcoTM, catalog number: 10270 )
  33. 2-Methylbutane (AKA Isopentane) (Sigma-Aldrich, catalog number: 277258 )
  34. Ethanol working solution (see Recipes)
  35. Paraformaldehyde (PFA) working solution (see Recipes)
  36. Sucrose working solutions (see Recipes)
  37. Cardiotoxin (CTX) 50 µM stock solution (see Recipes)
  38. Sterile phosphate-buffered saline (PBS) (see Recipes)
  39. 0.1% (1 mg/ml) pronase solution in serum-free DMEM (see Recipes)
  40. Media (see Recipes)
    1. 10% HS in DMEM
    2. 20% FBS in DMEM

Equipment

  1. 20 to 1,000 µl precision micropipettes
  2. Pipet controller
  3. Gas anesthesia systems: Isofluorane vaporizer, oxygen concentrator, flowmeter, induction chamber and gas mask (2B–2biological Instruments)
  4. Tissue culture laminar flow hood
  5. Surgical instruments:
    1. Fine scissors tungsten carbide straight 11.5 cm (Fine Science Tools, catalog number: 14568-12 )
    2. Fine scissors straight 10.5 cm (Fine Science Tools, catalog number: 14060-10 )
    3. Graefe forceps curved serrated (Fine Science Tools, catalog number: 11051-10 )
  6. Hamilton repeating dispenser, composed by the assembly of the three components shown in Figure 1:
    1. Hypodermic needle G30, 0.30 x 12.7 mm (PIC solution, catalog number: 03070300300.800 )
    2. Hamilton 50 μl 1705 LT syringe (Hamilton, catalog number: 80901 )
    3. PB600-1 repeating dispenser (Hamilton, catalog number: 83700 )
  7. 37 °C orbital shaker
  8. Centrifuge (we used a Beckman Coulter, model: Allegra X-22R centrifuge with SX4250 (Beckman Coulter, model: SX4250 ) and F2402H (Beckman Coulter, model: F2402H ) rotors for 15 ml and microcentrifuge tubes, respectively)
  9. Incubator for cell culture
  10. Hemocytometer chamber (Burker chamber or similar)
  11. Pannoramic Midi II automatic digital slide scanner (3DHISTECH, model: Pannoramic MIDI II )
  12. Fluorescence microscope/fluorescence slide scanner
  13. Confocal microscope (we used the Leica Microsystems, model: Leica TCS SP2 )
  14. Analytical balance
  15. Cryostat (we used the Leica Microsystems, model: Leica CM1850 UV )
  16. Autoclave


    Figure 1. Components to assemble a Hamilton repeating dispenser: hypodermic needle (A), Hamilton syringe (B), and repeating dispenser (C)

Software

  1. Image processing software such as ImageJ (https://imagej.net/)
  2. SPSS v17.0 software for Windows (SPSS; Chicago, IL)

Procedure

Note: All the procedures have been approved by the Institutional Animal Care and Use Committee at the University of Piemonte Orientale (Italy).

  1. Day -1: Damage induction in recipient muscles (Video 1)

    Video 1. Preparation of the recipient muscle. After removing the hairs from the front of the limb, cardiotoxin (CTX) is injected in the mid-belly region of the tibialis anterior (TA) muscle of the anesthetized mouse using a Hamilton repeating dispenser.

    1. Under sterile conditions, prepare 10 µM CTX from 50 µM stock solution in physiological solution (0.9% NaCl) and store on ice (see Recipes).
    2. Anesthetize the recipient mouse through isofluorane inhalation.
    3. Shave the hindlimbs by tearing off the hair with a pair of scissors to expose the tibialis anterior (TA) muscle (injection site) and clean the shaved skin with 70% ethanol (Video 1).
    4. Fill the Hamilton repeating dispenser (see Equipment for details) with CTX and inject it in 10 different sites (3 µl each) in the tibialis anterior mid-belly region (total volume injected: 30 µl) as shown in Video 1. The optimal positioning of injection sites is shown in Figure 2. After each CTX injection, leave the needle inside the muscle for at least 5 sec to avoid any leakage from the injection site. Special care should be taken when injecting in TA to avoid injecting beyond the thickness of the muscle.
    5. Clean again the shaved skin with 70% ethanol.
    6. Place back the mouse in its cage.


      Figure 2. Optimal positioning of the 10 CTX injection sites (white dots) in the TA muscle

  2. Day 0: Satellite cell isolation and transplantation (Videos 2 and 3)

    Video 2. Collection of muscles for SC isolation. After skin removal, hindlimb muscles and diaphragm are harvested from the GFP donor mouse and collected in pre-warmed serum-free DMEM.

    Video 3. Processing of muscles for SC isolation. An example of tendon removal (Achilles tendon removal from the gastrocnemius) and muscle processing for an optimal yield of SC isolation.

    The following steps describe the procedure to isolate SCs from a single donor mouse. The number of SCs obtained from a single donor is approximately 600,000 and is sufficient to transplant 5-6 mice.
    1. Pre-warm 10 ml serum-free DMEM and 10 ml PBS (see Recipes).
    2. Freshly prepare 3 ml 0.1% pronase solution in serum-free DMEM (see Recipes); under a biological hood, filter the pronase solution with a 0.2 µm microfilter in a 15 ml conical tube. For multiple preparations (i.e., SC isolation from multiple animals), use a separate tube for each animal to allow an optimal dissociation.
    3. Anesthetize a 10-week-old male GFP mouse through isofluorane inhalation and sacrifice by cervical dislocation.
    4. Soak the mouse for 2 min in 70% ethanol.
    Note: Perform the next steps under a biological hood.
    1. Fix the mouse to a support (e.g., with needles to a polystyrene box lid) and, with sterile surgical instruments (forceps and scissors), harvest the following muscles from both hindlimbs: tibialis anterior, extensor digitorum longus, quadriceps, gastrocnemius, soleus, and diaphragm (see Video 2 for details). Place the harvested muscles immediately in a 100 mm dish containing 10 ml of pre-warmed serum-free DMEM. Pre-wash the diaphragm in sterile PBS to remove the excess of blood and clots before adding it to the DMEM-containing dish.
      Note: During this step, it is required to remove the deep fascia (connective tissue that surrounds the muscles, shown in Figure 3) from each muscle to reduce fibroblast content during satellite cell isolation.


      Figure 3. Removal of the deep fascia. The arrows indicate the sheet of connective tissue surrounding the muscle.

    2. Place the muscles in a dish-cover (without medium) and remove tendons by using a sterile surgical blade (in Video 3, removal of gastrocnemius tendon in detail).
    3. Cut the processed muscles with a new sterile surgical blade into small pieces (about 1 mm3). During this step, add some drops of serum-free DMEM to prevent muscle from drying.
    4. Transfer the pieces of muscle into the tube containing the filtered 0.1% pronase solution. Place the tube on an orbital shaker at 37 °C for 60 min at 60 rpm.
    5. Centrifuge at 400 x g for 5 min at RT to collect and discard the supernatant without disturbing the pellet.
    6. Perform two cycles of mechanical trituration, which allows satellite cell release from the bulk in solution, as follows:
      1. Add 5 ml of pre-warmed 10% HS DMEM and disrupt the bulk by passing it several times (20-25) through a 10 ml serological pipette; leave the biggest fragments to settle on the bottom (for about 30 sec) and then carefully transfer the supernatant, containing the released cells, into a new 50 ml conical tube.
      2. Resuspend the remaining bulk of point (a) in 5 ml 10% HS DMEM and repeat the previous procedure in (Step B10a) by using a 5 ml (instead of 10 ml) serological pipette. Transfer the supernatant, containing the released cells, into the same 50 ml conical tube of point (Step B10a).
    7. Filter the pooled supernatant (10 ml) through a 40 µm cell-strainer into a new 50 ml conical tube. Add additional 5 ml of 10% HS DMEM to increase the yield of isolated cells.
    8. Centrifuge the cell suspension at 400 x g for 10 min at RT. Discard the supernatant and resuspend the pellet (isolated cells) with 10 ml of 20% FBS DMEM.
    9. Plate the resuspended cells in a 100 mm Petri dish and leave it undisturbed in the incubator (5% CO2, 37 °C) for 90 min. This pre-plating step allows fibroblast removal and satellite cell enrichment (fibroblasts will adhere to the Petri dish, while satellite cells will float in the medium).
    10. Collect the supernatant in a 15 ml conical tube and centrifuge it at 400 x g for 10 min at RT.
    11. Discard the supernatant without disturbing the pellet and resuspend the cells in 1 ml of 20% FBS DMEM, then transfer the cell suspension into a sterile 1.5 ml microcentrifuge tube.
    12. Count the round-shaped cells with a hemocytometer chamber or similar, excluding erythrocytes and debris.
    13. Centrifuge at 400 x g for 5 min at RT the 1.5 ml tube in a microcentrifuge, then discard the supernatant and carefully resuspend the cells in serum-free DMEM to a concentration of 4 x 106 cells/ml (i.e., 100,000 cells/25 µl) (Notes 1 and 2).
    14. Anesthetize recipient mice (as in Steps A2) and clean the region to be injected with 70% ethanol.
    15. Thoroughly mix the cell suspension and load the Hamilton repeating dispenser by removing any air bubble. Inject a total number of 100,000 cells (25 µl) in 5 different sites (5 µl each) in the mid-belly region of the pre-injured TA. The optimal positioning of injection sites is shown in Figure 4. Injections are performed as described in Video 1. After each injection of 5 µl cell suspension, leave the needle inside the muscle at the injection site for at least 5 sec. Clean the injected site with 70% ethanol.
    16. Place back the mouse in its cage.


      Figure 4. Optimal positioning of injection sites for GFP-SCs. Yellow dots indicate the 5 injection sites in pre-injured TA muscle.

  3. Day 30: Muscle harvesting and histological analysis
    1. For each muscle, prepare 500 µl 4% paraformaldehyde solution in a 1.5 ml microcentrifuge tube.
    2. Anesthetize the recipient mouse through isofluorane inhalation and sacrifice it by cervical dislocation.
    3. Fix the mouse (e.g., with needles to a polystyrene box lid) and harvest the transplanted TA muscle. Immerse the harvested muscle in 4% paraformaldehyde solution and leave it at 4 °C for 120 min to fix it.
      Note: This step is essential to avoid the solubilization (and loss) of GFP during the optional immunofluorescence procedure.
    4. Transfer the fixed muscle into a new 1.5 ml microcentrifuge tube containing 500 µl of 15% sucrose solution. Leave overnight at 4 °C to dehydrate the tissue.
    5. Continue muscle dehydration by transferring the muscle into a new 1.5 ml microcentrifuge tube containing 500 µl of 30% sucrose solution. Leave for 48 h at 4 °C.
    6. Carefully remove the muscle from the 30% sucrose solution, gently dab it on blotting paper, freeze it in liquid nitrogen-cooled isopentane, and store at -80 °C.
    7. Cut with a cryostat the frozen muscle in the mid-belly region (transplantation site) to obtain 7 µm-thick slices and assess the GFP-positive myofiber content through extemporaneous analysis under the fluorescent microscope.

Note: To increase the GFP signal, or if needed to co-investigate the expression of other proteins, proceed with the following immunofluorescence procedure.

  1. Immunofluorescence
    1. Post-fix the slices with 300 μl 4% paraformaldehyde on ice for 10 min.
    2. Wash 2 x 2 min with 300 μl PBS at RT.
    3. Permeabilize 1 x 5 min and 1 x 15 min with 300 μl 1% BSA-0.2% Triton X-100.
    4. Block with 300 μl 4% BSA for 30 min.
    5. Incubate for 2 h at RT (or overnight at 4 °C) with anti-GFP primary antibody diluted 1:500 in 100 μl 4% BSA.
    6. Wash 2 x 5 min and 2 x 15 min with 300 μl 1% BSA-0.2% Triton X-100.
    7. Incubate for 45 min with anti-rabbit secondary antibody diluted 1:400 in 100 μl 4% BSA.
    8. Wash 2 x 5 min and 2 x 15 min with 300 μl 0.2% Triton X-100.
    9. If identification of nuclei is needed, incubate for 5 min with 0.2 µg/ml DAPI in 300 μl PBS.
    10. Wash 2 x with 300 μl PBS for 2 min at RT.
    11. Mount the slices with a drop (~10 μl) of glycerol-based mounting medium.
    12. Acquire the images for the analysis (Figure 5). The whole muscle slice should be acquired, for example by using an instrument such as the Pannoramic Midi II (3DHISTECH).


      Figure 5. Representative image of GFP+ fibers derived from transplanted GFP+ SCs. Image obtained with the Leica SP2 confocal microscope of a tibialis anterior transversal section of a recipient mouse 30 days after transplantation of SCs from a GFP donor mouse. The slice has been stained with the anti-GFP primary and 488-conjugated secondary antibodies to amplify the GFP signal, following Procedure D.

    The evaluation of transplantation efficacy among different experimental conditions (such as the comparative analysis between wild-type and transgenic or knockout recipient mice) requires a quantification of the number of the GFP-positive myofibers in the whole muscle slice. A myofiber is scored as ‘GFP-positive’ when the fluorescent intensity significantly differs from the background. A considerable variability in GFP intensity is expected, depending on the number of GFP-positive SCs fused to the fiber. For an unbiased quantification and to increase data reproducibility, use an image processing software such as ImageJ (https://imagej.net/) as follows:
    1. Open ImageJ software;
    2. Open the image to be analyzed (File > Open);
    3. Set the background completely under the threshold (Image > Adjust > Threshold; Figure 6)


      Figure 6. GFP-positive fibers quantification with ImageJ. The original fluorescence image (upper panel) is processed by setting a threshold limit, clearly defining a black background and the GFP-positive myofibers displayed in red (lower panel).

    Apply the same setting for all the images in the different experimental groups and quantify the number of GFP-positive myofibers (i.e., myofibers with a fluorescence intensity higher than the threshold).
    In Figures 1F and 1G of the original paper (Reano et al., 2017), the results refer to the SC transplantation from 3 GFP donor mice in 11 C57BL/6J wild-type recipient mice (Mean number of GFP-positive myofibers = 39.45; standard deviation = 22.15; standard error = 7.00).

Data analysis

To compare two data sets, as in Reano et al., 2017, in which we assessed the effect of high levels of unacylated ghrelin in SC engraftment, we performed Mann U Whitney test with the SPSS v17.0 software for Windows (SPSS; Chicago, IL). Statistical significance was assumed for P < 0.05.

Notes

  1. Each recipient muscle will be injected with 100,000 cells in a volume of 25 µl, but a total volume of 75 µl should be considered because of the Hamilton repeating dispenser dead volume.
  2. Usually, the number of satellite cells obtained from a single donor mouse is approximately 600,000.

Recipes

  1. Ethanol working solution
    70% v/v ethanol in deionized water
  2. Paraformaldehyde (PFA) working solution
    4% w/v PFA in PBS
  3. Sucrose working solutions
    15% or 30% w/v sucrose in deionized water
  4. Cardiotoxin (CTX) 50 µM stock solution
    Dissolve 1 mg of CTX in 2.94 ml 0.9% NaCl
    Aliquot and store at -20 °C
    Avoid freeze-thaw cycles
    Note: Prepare the 10 µM working solution (in 0.9% NaCl) just before use.
  5. Sterile phosphate-buffered saline (PBS)
    137 mM sodium chloride (NaCl)
    2.7 mM potassium chloride (KCl)
    10 mM sodium phosphate dibasic heptahydrate (Na2HPO4·7H2O)
    1.8 mM potassium phosphate monobasic (KH2PO4)
    To prepare 1 L of PBS, dissolve 8 g NaCl, 200 mg KCl, 1.15 g Na2HPO4·7H2O, and 240 mg KH2PO4 in ultrapure water and adjust the pH to 7.4; sterilize the solution by autoclaving
  6. 0.1% (1 mg/ml) pronase solution in serum-free DMEM
    The volume required is 3 ml for each donor GFP mouse (3 mg of pronase powder); however, we recommend to weigh 10 mg of powder in 1 ml (1% pronase solution), then dilute it tenfold to make the 0.1% pronase working solution. Filter the pronase solution with a 0.2 µm microfilter in sterile conditions
  7. Media
    Supplement 495 ml of DMEM with 5 ml of antibiotic-antimycotic solution
    To prepare 15 ml of 10% HS in DMEM: add 1.5 ml of HS to 13.5 ml of DMEM supplemented with antibiotic-antimycotic; store at 4 °C; pre-warm before use
    To prepare 15 ml of 20% FBS in DMEM: add 3 ml of FBS to 12 ml of DMEM supplemented with antibiotic-antimycotic; store at 4 °C; pre-warm before use

Acknowledgments

The satellite cell isolation procedure has been adapted from Danoviz et al., 2012 and Musarò et al., 2010; the SC transplantation procedure was adapted from Liu et al., 2012.
This study was supported by research grant from the Muscular Dystrophy Association (grant No. MDA294617 to NF and AG) and Fondazione Cariplo (2015-0634 to NF).
AG is a consultant to Helsinn (Lugano, Switzerland); NF is a consultant to Lyric Pharmaceuticals (South San Francisco, CA, US).
Authors declare that they have no conflicts of interest or competing interests.

References

  1. Collins, C. A., Olsen, I., Zammit, P. S., Heslop, L., Petrie, A., Partridge, T. A. and Morgan, J. E. (2005). Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell 122(2): 289-301.
  2. Danoviz, M. E. and Yablonka-Reuveni, Z. (2012). Skeletal muscle satellite cells: background and methods for isolation and analysis in a primary culture system. Methods Mol Biol 798: 21-52.
  3. Liu, W., Wen, Y., Bi, P., Lai, X., Liu, X. S., Liu, X. and Kuang, S. (2012). Hypoxia promotes satellite cell self-renewal and enhances the efficiency of myoblast transplantation. Development 139(16): 2857-2865.
  4. Mauro, A. (1961). Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol 9: 493-495.
  5. Musarò, A. and Barberi, L. (2010). Isolation and culture of mouse satellite cells. Methods Mol Biol 633: 101-111.
  6. Partridge, T. (2002). Myoblast transplantation. Neuromuscul Disord 12 Suppl 1: S3-6.
  7. Partridge, T. A., Grounds, M. and Sloper, J. C. (1978). Evidence of fusion between host and donor myoblasts in skeletal muscle grafts. Nature 273: 306-308.
  8. Reano, S., Angelino, E., Ferrara, M., Malacarne, V., Sustova, H., Sabry, O., Agosti, E., Clerici, S., Ruozi, G., Zentilin, L., Prodam, F., Geuna, S., Giacca, M., Graziani, A. and Filigheddu, N. (2017). Unacylated ghrelin enhances satellite cell function and relieves the dystrophic phenotype in duchenne muscular dystrophy mdx model. Stem Cells 35(7): 1733-1746.

简介

卫星细胞(SC)移植代表了肌肉再生期间SC生物学研究的强大策略。 我们在这里描述了从绿色荧光蛋白(GFP)表达的小鼠和他们的小鼠肌肉移植SC分离的协议。 该程序最初用于评估激素非酰化生长素释放肽对肌肉再生的影响,特别是评估受体肌肉中未酰化的生长素释放肽的增加如何影响供体SC的植入(Reano等人,2017年)。

【背景】由分化的肌纤维组成的骨骼肌可以在受伤时再生。肌肉再生依赖于居住在肌肉纤维基底层之下的称为卫星细胞(SC)的静止驻留干细胞群(Mauro,1961)。受伤后,SC经历激活,广泛的增殖,分化和融合,最终修复或替换受损的肌纤维(Collins等人,2005)。

由于移植的成肌细胞可以与宿主成肌细胞融合,因此SC的移植被认为是杜氏肌营养不良症(DMD)的潜在疗法多年的时间,这提示了在缺陷纤维中功能性修复的可能性(Partridge 等,1978)。不幸的是,在DMD患者的人体临床试验中,该策略未能使注射肌肉中的肌营养不良蛋白得以恢复,并且没有观察到功能改善(Partridge,2002)。尽管如此,SCs移植仍然是SC生物学研究的一个强有力的策略,主要研究细胞自主和非细胞自主的肌肉再生机制。在这个协议中,我们描述了一种从GFP表达的供体小鼠中移出分离的细胞的方法,其允许在受体肌肉中容易地追踪和测量移植的细胞。我们使用了一种简单且相对便宜的细胞分离方法;然而,要移植的细胞可以通过不同的技术分离,如荧光激活细胞分选(FACS)或磁珠。

关键字:卫星细胞, 肌肉干细胞, 移植, 植入, 骨骼肌, 再生, 肌肉切除, 心脏毒素

材料和试剂

  1. 0.2微米微型过滤器(VWR,目录号:28145-475)
  2. 一次性无菌塑料:
    1. 15毫升锥形管
    2. 100毫米培养皿
    3. 10毫升锥形管
    4. 5毫升血清移液器
    5. 10毫升血清移液器
    6. 1.5毫升微量离心管
    7. 20到1,000微升移液枪头
    8. 10毫升塑料注射器(Terumo Medical,产品目录号:SS-10L)
  3. 外科刀片(Moretti S.p.a.-Chimo)
  4. 40微米细胞过滤器(Corning,目录号:431750)
  5. 粘附显微镜载玻片Superfrost Ultraplus(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:J3800AMNZ)
  6. 22 x 22毫米幻灯片盖玻片(Bio-Optica,目录号:09-02222)
  7. 羽毛切片机C35刀片(PFM Medical,目录号:207500003)
  8. 一般用途的吸墨纸
  9. 10周龄的C57BL / 6-Tg(CAG-EGFP)131Osb / LeySopJ小鼠(GFP小鼠)作为供体小鼠(THE JACKSON LABORATORY,目录号:006567)
  10. 4个月大的C57BL / 6J,作为受体小鼠(THE JACKSON LABORATORY,目录号:000664)
  11. 异氟烷(Isofluorane-vet,Merial Italia,目录号:00018152)
  12. 乙醇(Honeywell International,目录号:458600)
  13. 液氮
  14. 低温包埋介质,Killik(Bio-Optica,目录号:05-9801)
  15. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2153)
  16. Triton X-100(Sigma-Aldrich,目录号:T9284)
  17. 兔抗绿色荧光蛋白(GFP)抗体(Thermo Fisher Scientific,Invitrogen,目录号:A-11122)
  18. 驴抗兔IgG(H + L)Alexa Fluor 488结合物(Thermo Fisher Scientific,Invitrogen,目录号:A-21206)
  19. 4',6-二脒基-2-苯基吲哚二盐酸盐(DAPI)(Thermo Fisher Scientific,Invitrogen TM,目录号:D1306)
  20. 缓慢褪色,基于甘油的固定介质(Thermo Fisher Scientific,Invitrogen TM,目录号:S36936)
  21. 多聚甲醛(PFA)(Sigma-Aldrich,目录号:P6148)
  22. 蔗糖(Sigma-Aldrich,目录号:S0389)
  23. 来自Naja pallida的心脏毒素(CTX)(Latoxan,目录号:L8102)
  24. 0.9%NaCl(Sigma-Aldrich,目录号:S8776)
  25. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S3014)
  26. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333)
  27. 磷酸二氢钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:255793)
  28. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P5655)
  29. 链霉菌Streptomyces griseus的链霉蛋白酶(Sigma-Aldrich,Roche Diagnostics,目录号:10165921001)
  30. 用抗生素 - 抗霉菌溶液(Sigma-Aldrich,目录号:A5955)补充(对于所有操作)的DMEM-Dulbecco改良的Eagle培养基,高葡萄糖(Thermo Fisher Scientific,Gibco TM,目录号:11965092)
  31. 供体马血清(HS)(Thermo Fisher Scientific,Gibco TM,目录号:16050130)
  32. 胎牛血清,FBS(Thermo Fisher Scientific,Gibco,产品目录号:10270)
  33. 2-甲基丁烷(AKA异戊烷)(Sigma-Aldrich,目录号:277258)
  34. 乙醇工作液(见食谱)
  35. 多聚甲醛(PFA)工作溶液(见食谱)
  36. 蔗糖工作解决方案(见食谱)
  37. 心脏毒素(CTX)50μM储备液(见食谱)
  38. 无菌磷酸盐缓冲盐水(PBS)(见食谱)
  39. 0.1%(1毫克/毫升)链霉蛋白酶溶液在无血清DMEM(见食谱)
  40. 媒体(见食谱)
    1. DMEM中10%的HS
    2. DMEM中20%FBS

设备

  1. 20至1,000微升精密微量移液器
  2. 移液器控制器
  3. 气体麻醉系统:异氟醚蒸发器,氧气浓缩器,流量计,感应室和防毒面具(2B-2生物仪器)
  4. 组织培养层流罩
  5. 手术器械:
    1. 精细剪刀硬质合金直线11.5厘米(Fine Science Tools,目录号:14568-12)
    2. 精细剪刀直10.5厘米(精细科学工具,目录号:14060-10)
    3. Graefe钳弯曲的锯齿(Fine Science Tools,目录号:11051-10)
  6. 汉密尔顿重复分配器,由图1所示的三个组件组成:
    1. 皮下注射针G30,0.30 x 12.7毫米(PIC解决方案,目录号:03070300300.800)
    2. Hamilton 50μl1705 LT注射器(Hamilton,目录号:80901)
    3. PB600-1重复分配器(汉密尔顿,目录号:83700)
  7. 37°C轨道摇床
  8. 离心机(我们使用Beckman Coulter,型号为:Allegra X-22R离心机,SX4250(Beckman Coulter,型号:SX4250)和F2402H(Beckman Coulter,型号:F2402H)转子分别用于15ml和微量离心管)。
  9. 培养细胞培养
  10. 血细胞计数器室(Burker室或类似)
  11. Pannoramic Midi II自动数字滑盖扫描仪(3DHISTECH,型号:Pannoramic MIDI II)
  12. 荧光显微镜/荧光幻灯片扫描仪
  13. 共聚焦显微镜(我们使用徕卡显微系统,型号:徕卡TCS SP2)
  14. 分析天平
  15. Cryostat(我们使用徕卡显微系统,型号:Leica CM1850 UV)
  16. 高压灭菌器


    图1.组装汉密尔顿重复分配器的组件:皮下注射针(A),汉密尔顿注射器(B)和重复分配器(C)

软件

  1. ImageJ等图像处理软件( https://imagej.net/
  2. 用于Windows的SPSS v17.0软件(SPSS; Chicago,IL)

程序

注意:所有程序均已获得意大利Piemonte Orientale大学动物管理和使用委员会的批准。

  1. 第一天:受伤肌肉受损(视频1)

    视频1

    1. 在无菌条件下,从生理溶液(0.9%NaCl)中的50μM储备液中制备10μMCTX并储存在冰上(参见食谱)。
    2. 通过异氟醚吸入麻醉受体小鼠。

    3. 用一把剪刀剪掉头发,以暴露胫骨前肌(TA)肌肉(注射部位)并用70%乙醇清洁剃光的皮肤(视频1),从而剃去后肢。
    4. 用CTX填充汉密尔顿重复分配器(详见设备),并在视频1中所示的胫骨前中腹部区域(注射总体积:30μl)中的10个不同位点(每个3μl)注射。的注射部位如图2所示。每次CTX注射后,将针头留在肌肉内至少5秒以避免注射部位的任何泄漏。在TA注射时应特别小心,避免注射超出肌肉厚度。
    5. 用70%乙醇再次清洁剃光的皮肤。
    6. 把鼠标放在笼子里。


      图2. TA肌肉中10个CTX注射部位(白点)的最佳位置

  2. 第0天:卫星细胞隔离和移植(视频2和3)

    视频2

    视频3

    以下步骤描述了从单个供体小鼠分离SC的程序。从一个捐助者获得的SC数量约为600,000,足以移植5-6只小鼠。
    1. 预热10毫升无血清DMEM和10毫升PBS(见食谱)。
    2. 在无血清DMEM中新鲜制备3ml 0.1%链霉蛋白酶溶液(参见食谱);在生物罩下,用0.2μm微滤器在15ml锥形管中过滤链霉蛋白酶溶液。对于多种制剂(即从多种动物中分离SC),对每只动物使用单独的管以允许最佳的解离。
    3. 麻醉一个10周龄的雄性GFP小鼠,通过吸入异氟烷并颈椎脱臼处死。
    4. 在70%乙醇中浸泡2分钟。
    注意:在生物罩下进行下一步。
    1. 将鼠标固定在支架上(例如,用针头固定在聚苯乙烯盒盖上),用无菌手术器械(钳子和剪刀)从两个后肢收获以下肌肉:胫骨前部,趾长伸肌,股四头肌,腓肠肌,比目鱼肌和膈肌(详见视频2)。将收获的肌肉立即放入含有10ml预热的无血清DMEM的100mm培养皿中。在无菌PBS中预先清洗隔膜以除去多余的血液和凝块,然后将其加入到含有DMEM的培养皿中。
      注意:在此步骤中,需要从每个肌肉中去除深筋膜(图3所示的围绕肌肉的结缔组织)以减少卫星细胞分离期间的成纤维细胞含量。


      图3.去除深筋膜箭头指示肌肉周围的结缔组织片。

    2. 将肌肉放在盖板(无中等)中,使用无菌手术刀(视频3,详细切除腓肠肌肌腱)去除肌腱。
    3. 用新的无菌手术刀将加工过的肌肉切成小块(约1毫米×3毫米)。在这个步骤中,加入一些无血清的DMEM,以防止肌肉干燥。
    4. 将肌肉块转移到含有过滤的0.1%链霉蛋白酶溶液的管中。将试管置于37℃的定轨摇床上60rpm。
    5. 在室温下400×g离心5分钟以收集并丢弃上清液而不干扰沉淀。
    6. 进行两个机械研磨循环,这允许卫星细胞从溶液中散装出来,如下所述:
      1. 加入5毫升预热的10%HS DMEM,并通过10毫升血清移液管将其通过几次(20-25)破碎。留下最大的碎片沉降在底部(约30秒),然后小心地将含有释放的细胞的上清液转移到新的50ml锥形管中。
      2. 用5毫升10%HS DMEM重新悬浮点(a)的剩余部分,并使用5毫升(而不是10毫升)血清移液管重复(步骤B10a)中的前述程序。将含有释放的细胞的上清液转移到同一个点(步骤B10a)的50ml锥形管中。
    7. 过滤收集的上清液(10毫升)通过一个40微米细胞过滤器到一个新的50毫升锥形管。再加入5ml的10%HS DMEM以增加分离细胞的产量。
    8. 在室温下将细胞悬浮液在400×g下离心10分钟。弃去上清,用10ml 20%FBS DMEM重悬沉淀(分离的细胞)。
    9. 将重新悬浮的细胞铺板于100mm培养皿中,并使其在培养箱(5%CO 2,37℃)中静置90分钟。这个预镀步骤允许去除成纤维细胞和卫星细胞富集(成纤维细胞将粘附在培养皿上,而卫星细胞将漂浮在培养基中)。
    10. 将上清液收集在15ml锥形管中并在室温下以400×g离心10分钟。
    11. 弃去上清液而不干扰沉淀,并将细胞重悬于1ml 20%FBS DMEM中,然后将细胞悬浮液转移到无菌的1.5ml微量离心管中。
    12. 用血细胞计数器或类似物计数圆形细胞,排除红细胞和碎片。
    13. 在400×g离心5分钟,1.5ml离心管中,弃去上清液,小心地将细胞重悬于无血清的DMEM中,浓度为4×10-6, (ml),100,000个细胞/25μl)(注1和2)。
    14. 麻醉受体小鼠(如步骤A2),并用70%乙醇清洁要注射的区域。
    15. 彻底混合细胞悬液,并通过消除气泡来加载汉密尔顿重复分配器。在伤前TA的中腹部区域,在5个不同位点(每个5μl)注射总计100,000个细胞(25μl)。注射部位的最佳位置如图4所示。按照视频1中所述进行注射。每次注射5μl细胞悬液后,将注射部位的肌肉留在肌肉内至少5秒。用70%乙醇清洗注射部位。
    16. 把鼠标放在笼子里。


      图4. GFP-SC注射位置的最佳位置。
      黄点表示伤前TA肌肉中的5个注射部位。

  3. 第30天:肌肉采集和组织学分析
    1. 对于每个肌肉,准备500毫升4%多聚甲醛溶液在1.5毫升微量离心管。
    2. 通过吸入异氟烷麻醉受体小鼠,并通过颈椎脱臼将其牺牲。
    3. 将鼠标(,例如,用针头固定在聚苯乙烯盒盖上)固定并收获移植的TA肌肉。将收获的肌肉浸入4%多聚甲醛溶液中,4℃放置120分钟固定。
      注意:这一步对于避免GFP在选择性免疫荧光过程中的溶解(和损失)是必不可少的。
    4. 将固定的肌肉转移到含有500μl15%蔗糖溶液的新的1.5ml微量离心管中。
      在4°C放置过夜使组织脱水
    5. 通过将肌肉转移到含有500μl30%蔗糖溶液的新的1.5ml微量离心管中继续肌肉脱水。
      在4°C放置48小时
    6. 小心地从30%的蔗糖溶液中取出肌肉,轻轻地将其浸在吸墨纸上,在液氮冷却的异戊烷中冷冻,并储存在-80°C。
    7. 用低温恒温器切下中腹部区域(移植部位)中的冷冻肌肉以获得7μm厚的切片并通过荧光显微镜下的即时分析评估GFP阳性肌纤维含量。

注意:为了增加GFP信号,或者如果需要共同研究其他蛋白质的表达,继续进行以下免疫荧光程序。

  1. 免疫荧光
    1. 用300μl4%多聚甲醛在冰上将所述切片后固定10分钟。

    2. 用300μlPBS在室温下洗2 x 2分钟
    3. 用300μl1%BSA-0.2%Triton X-100透化1×5分钟和1×15分钟。
    4. 用300μl4%BSA封闭30分钟。
    5. 在室温下孵育2小时(或在4℃过夜),用在100μl4%BSA中1:500稀释的抗GFP初级抗体。
    6. 用300μl1%BSA-0.2%Triton X-100洗2次5分钟和2次15分钟。

    7. 用100μl4%BSA中1:400稀释的抗兔二抗孵育45分钟
    8. 用300μl0.2%Triton X-100洗2次5分钟和2次15分钟。
    9. 如果需要对细胞核进行鉴定,用300μlPBS中的0.2μg/ ml DAPI孵育5分钟。

    10. 用300μlPBS洗涤2次,2分钟
    11. 用一滴(〜10微升)的甘油基安装介质装上切片。
    12. 获取图像进行分析(图5)。应该获得整个肌肉切片,例如通过使用诸如Pannoramic Midi II(3DHISTECH)的仪器。


      图5.来自移植的GFP + SC的GFP +纤维的代表性图像。在从GFP供体小鼠的SC移植30天后,用受体小鼠的胫骨前横切片的Leica SP2共焦显微镜获得的图像。按照程序D,将切片用抗GFP初级抗体和488-偶联的二级抗体染色以扩增GFP信号。

    不同实验条件(例如野生型和转基因或敲除受体小鼠之间的比较分析)之间的移植功效的评价需要定量整个肌肉切片中的GFP阳性肌纤维的数量。当荧光强度与背景显着不同时,肌纤维评分为“GFP阳性”。预计GFP强度有相当大的变化,这取决于与纤维融合的GFP阳性SC的数量。为了实现无偏差的量化并提高数据的可重复性,请使用ImageJ等图像处理软件( https://imagej.net/)如下:
    1. 打开ImageJ软件;
    2. 打开要分析的图像(文件&打开);
    3. 将背景完全设置在阈值(图像&gt;调整&gt;阈值;图6)


      图6.使用ImageJ对GFP阳性纤维进行定量通过设置阈值限制来处理原始荧光图像(上面板),明确定义黑色背景和GFP-正面肌纤维显示为红色(下面板)。

    对不同实验组的所有图像应用相同的设置,并量化GFP阳性肌纤维数量(即,荧光强度高于阈值的肌纤维)。
    在原始文章(Reano等人,2017)的图1F和1G中,结果涉及来自11只C57BL / 6J野生型受体小鼠的3只GFP供体小鼠的SC移植(平均数的GFP阳性肌纤维= 39.45;标准偏差= 22.15;标准误差= 7.00)。

数据分析

为了比较两个数据集,如2017年的Reano等人,其中我们评估了SC植入中高度未酰化的生长素释放肽的影响,我们使用SPSS v17.0进行Mann U Whitney检验Windows版软件(SPSS; Chicago,IL)。统计显着性假定为 0.05。

笔记

  1. 每个受体肌肉将被注入25000μl体积的100,000个细胞,但由于汉密尔顿重复的分配器死体积,应考虑总体积为75μl。
  2. 通常,从一个供体小鼠获得的卫星细胞数量大约是60万。

食谱

  1. 乙醇工作解决方案
    去离子水中70%v / v乙醇
  2. 多聚甲醛(PFA)工作溶液
    PBS中4%w / v PFA
  3. 蔗糖工作解决方案
    去离子水中15%或30%w / v蔗糖
  4. 心脏毒素(CTX)50μM储备液
    将1毫克CTX溶于2.94毫升0.9%NaCl中
    分装和存储在-20°C
    避免冻融循环
    注意:在使用前准备10μM工作溶液(在0.9%NaCl中)。
  5. 无菌磷酸缓冲盐水(PBS)
    137毫摩尔氯化钠(氯化钠)
    2.7 mM氯化钾(KCl)
    10mM磷酸氢二钠七水合物(Na 2 HPO 4•7H 2 O)
    1.8 mM磷酸二氢钾(KH 2 PO 4)
    为制备1L的PBS,溶解8g NaCl,200mg KCl,1.15g Na 2 HPO 4•7H 2 O和240 mg KH 2 PO 4 4在超纯水中调节pH至7.4;通过高压灭菌对溶液进行灭菌
  6. 0.1%(1毫克/毫升)的链霉蛋白酶溶液在无血清DMEM
    每个供体GFP小鼠(3mg链霉蛋白酶粉末)所需的体积为3ml;然而,我们建议在1毫升(1%链霉蛋白酶溶液)中称取10毫克粉末,然后稀释10倍,制成0.1%链霉蛋白酶工作溶液。用无菌条件下的0.2微米微滤器过滤链霉蛋白酶溶液
  7. 媒体
    补充495毫升的DMEM与5毫升的抗生素 - 抗真菌溶液
    在DMEM中制备15ml 10%HS:将1.5ml HS加入到13.5ml补充有抗生素 - 抗真菌剂的DMEM中;在4°C储存;预热前使用
    在DMEM中制备15ml 20%FBS:将3ml FBS加入到12ml补充有抗生素 - 抗真菌剂的DMEM中;在4°C储存;预热前使用

致谢

卫星细胞分离程序已经改编自Danoviz等人,2012和Musarò等人,2010; SC移植程序改编自Liu et al。,2012。
本研究由肌肉萎缩症协会(批准号MDA294617至NF和AG)和卡里(Fondazione Cariplo)(2015-0634至NF)的研究资助支持。
AG是Helsinn(瑞士卢加诺)的顾问; NF是Lyric制药公司(美国加利福尼亚州南旧金山)的顾问。
作者声明他们没有利益冲突或利益冲突。

参考

  1. Collins,C.A.,Olsen,I.,Zammit,P.S.,Heslop,L.,Petrie,A.,Partridge,T.A。和Morgan,J.E。(2005)。 干细胞功能,自我更新和来自成年肌肉卫星细胞生态位的细胞行为异质性。 Cell 122(2):289-301。
  2. Danoviz,M.E。和Yablonka-Reuveni,Z。(2012)。 骨骼肌卫星细胞:在原代培养体系中分离和分析的背景和方法
  3. Liu,W.,Wen,Y.,Bi,P.,Lai,X.,Liu,X.S。,Liu,X.和Kuang,S。(2012)。 缺氧促进卫星细胞自我更新并提高成肌细胞移植的效率 发展 139(16):2857-2865。
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引用:Angelino, E., Reano, S., Ferrara, M., Agosti, E., Sustova, H., Malacarne, V., Clerici, S., Graziani, A. and Filigheddu, N. (2018). Mouse Satellite Cell Isolation and Transplantation. Bio-protocol 8(2): e2696. DOI: 10.21769/BioProtoc.2696.
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