The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.
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Motohashi, N., Asakura, Y., Asakura, A. Isolation, Culture, and Transplantation of Muscle Satellite Cells. J. Vis. Exp. (86), e50846, doi:10.3791/50846 (2014).
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Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.
However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.
Muscle satellite cells are a small population of myogenic stem cells located beneath the basal lamina of skeletal muscle fibers. They are characterized by the expression of Pax7, Pax3, c-Met, M-cadherin, CD34, Syndecan-3, and calcitonin1-3. Satellite cells have proven to be responsible for muscle regeneration as muscle stem cells. In adult muscle, satellite cells are normally mitotically quiescent4-8. Following injury, satellite cells are activated, initiate expression of MyoD, and enter the cell cycle to expand their progeny, termed myogenic precursor cells or myoblasts3. After several rounds of cell division, myoblasts exit the cell cycle and fuse to each other in order to undergo differentiation into multi-nucleated myotubes, followed by mature muscle fibers. Myoblasts isolated from adult muscle can readily be expanded ex vivo. The capacity for myoblasts to become muscle fibers in regenerating muscle and to form ectopic muscle fibers in nonmuscle tissues is exploited by myoblast transplantation, a potential therapeutic approach for Duchenne muscular dystrophy (DMD)4, urological dysfunction9, and heart failure10. Indeed, myoblasts have been successfully transplanted in the muscle of both mdx (DMD model) mice and DMD patients11-14. The injected normal myoblasts fuse with host muscle fibers to improve the histology and function of the diseased muscle. Previous work demonstrated that subpopulations of myoblasts are more stem cell-like and remain in an undifferentiated state longer in muscle during muscle regeneration5. Recent work has shown that freshly isolated satellite cells from adult muscle contain a stem cell-like population that exhibits more efficient engraftment and self-renewal activity in regenerating muscle5-8. Therefore, purification of a pure population of quiescent satellite cells from adult skeletal muscle is essential for understanding the biology of satellite cells, myoblasts and muscle regeneration, and for the development of cell-based therapies.
However, current prospective purification methods of quiescent satellite cells require the use of an expensive fluorescence-activated cell sorting (FACS) machine1,2,6-8. In addition, FACS laser exposure tends to induce cell death during separation, which causes lower yield of quiescent satellite cells15. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle. This method utilizes enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. We also show that intramuscular injection of these freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.
The animals were housed in an SPF environment and were monitored by the Research Animal Resources (RAR) of the University of Minnesota. The animals were euthanized by appropriate means (CO2 inhalation or KCl injection after being anesthetized with IP injection of Avertin (250 mg/kg). All protocols were approved by the Institutional Animal Care and Use Committee (IACUC, Code Number: 1304-30492) of the University of Minnesota.
1. Isolation of Mononuclear Cells from Mouse Skeletal Muscle
- Properly sacrifice 1 or 2 young adult mice (3-8 weeks).
- Pinch and slit the skin of the abdomen with sharp scissors. Peel off skin to completely show triceps and hind limb muscle (pull the skin in opposing directions).
- Remove all leg skeletal muscles (tibialis anterior, gastrocnemius, and quadriceps) and triceps along the bones with scissors. Then transfer muscles to ice-cold, sterile PBS in a 10 cm plate.
- Wash blood off muscles in PBS and transfer muscles to a new sterile 6 cm plate: 1 plate for 1-2 mice.
- Remove connective tissue, blood vessels, nerve bundles, and adipogenic tissue under a dissection microscope.
- Using scissors for ophthalmology, cut and mince the tissue into a smooth pulp (Figures 1A and 1B). Try not to leave large pieces, as they will not be broken down readily by the enzyme solution.
- Transfer minced muscles into a Falcon 50 ml tube, and add 5 ml of collagenase solution (0.2% collagenase Type 2 in 10% FBS in DMEM). Incubate at 37 °C for 60 min.
- Triturate (up and down with an 18 G needle) to homogenize mixture (Figure 1C). Then further incubate the mixture at 37 °C for 15 min.
- Triturate again to homogenize mixture to dissociate into single cell suspension. Add 2% FBS in DMEM up to 50 ml into the single cell suspension and mix well.
- Place a cell strainer (70 μm) onto a Falcon 50 ml tube (Figure 1D). Transfer the supernatant containing the dissociated cells onto a cell strainer, and pipette the cell suspension up and down on the filter until it passes through.
- Count cell number by hemocytometer. Centrifuge the tubes at 2,000 rpm at 4 °C for 5 min; aspirate and discard the supernatant.
- Resuspend with 10 ml of 2% FBS in DMEM. Centrifuge the tubes at 2,000 rpm at 4 °C for 5 min; aspirate and discard the supernatant.
- Resuspend with 200 μl of 2% FBS in DMEM and transfer cell suspension into 1.5 ml microcentrifuge tubes. Usually, about 2 x 106 cells should be harvested from muscles of 1 mouse. Cells will be diluted to a concentration of 1 x 106 cells in 100 μl of 2% FBS in DMEM.
2. Antibody Staining and Separation with MACS
During the following procedures, maintain sterile conditions by using sterile buffers. Each volume of antibodies added and cell suspension medium is calculated for cells from whole muscles of 1 mouse. If cells are harvested from 2 or more mice, the amount of reagents should be optimized.
- Add 1 μl each of CD31-PE, CD45-PE, Sca-1-PE, and Integrin α7 antibody into 200 μl of the cell suspension. Incubate on ice for 30 min.
- Wash cells: After incubation, add 1 ml of 2% FBS in DMEM into the cell suspension in the 1.5 ml tube, and centrifuge at 2,000 rpm at 4 °C for 3 min. Repeat this step twice.
- Aspirate and discard the supernatant.
- Resuspend cells with 200 μl of 2% FBS in DMEM, and add 10 μl of Anti-PE Magnetic Beads. Incubate on ice for 30 min.
- Wash cells: After incubation, add 1 ml of MACS buffer to the cell suspension in the 1.5 ml microcentrifuge tube, and then centrifuge at 2,000 rpm at 4 °C for 3 min. Repeat this step twice. Note that cells should be washed by MACS buffer before being separated by magnetic column.
- Aspirate and discard the supernatant. Resuspend the cells with 1.0 ml of MACS buffer.
- Set up LD column on a Magnetic board, and rinse the column with 2.0 ml of MACS buffer (Figure 1E).
- Transfer the cell suspension onto the LD column, and collect the flow-through fraction into a 1.5 ml tube. This fraction contains PE-negative cells.
- Centrifuge at 2,000 rpm at 4 °C for 3 min, aspirate and discard the supernatant.
- Resuspend cells with 200 μl of 2% FBS in DMEM, and add 10 μl of Anti-Mouse IgG Magnetic Beads. Incubate on ice for 30 min.
- Wash cells: After incubation, add 1 ml of MACS buffer into the cell suspension in 1.5 ml tube, and then centrifuge at 2,000 rpm at 4 °C for 3 min. Aspirate and discard the supernatant. Repeat this step twice. Resuspend cells with 500 μl of MACS buffer.
- Set up MS column on a Magnetic board, and rinse the column with 500 μl of MACS buffer (Figure 1F).
- Transfer suspended cell solution onto the MS column, and discard the flow-through fraction (Integrin α7-negative cells).
- Rinse with 1 ml of MACS buffer, and repeat this step twice.
- After rinsing, remove column from the magnetic field of separator. Apply 1.0 ml of MACS buffer onto the column, and elute the magnetically labeled cells (Integrin α7-positive cells) into a 1.5 ml microcentrifuge tube by pushing the syringe plunger from the top of the column. Collect the flow-through into a 1.5 ml tube. Repeat the elution with 1.5 ml of MACS buffer and collect the flow-through.
- Centrifuge at 2,000 rpm at 4 °C for 3 min, aspirate and discard the supernatant.
- Resuspend purified cells with 1 ml of Myoblast medium (20% FBS contained Hams F-10 with bFGF), and plate cells on Matrigel-coated 10 cm plate with 8 ml of Myoblast Medium (5 ml in 6 cm plate) (Figure 1G). Note that 1-2 x 105 cells can potentially be isolated from 1 intact mouse muscle.
- Feed cells every other day with Myoblast medium. The appearance of growing myoblasts is of a small and round shape expressing MyoD (Figure 2) and Pax7 (data not shown) in their nucleus.
- Myoblasts should be passaged before 50% confluence or when starting cell fusion. After rinsing once with PBS, incubate cells with 0.25% Trypsin solution at 37 °C for 3 min in a CO2 incubator and collect dissociated cells with Myoblast medium. After centrifuge cells (1,000 rpm for 5 min), suspend with Myoblast medium and replate cells onto new Matrigel-coated plates. Collagen-coated plates can be used after passage 3. One plate can usually be split into three to five plates.
- Refeed with Differentiation Medium every other day.
- By day 1 in Differentiation Medium, myoblasts exit the cell cycle and undergo differentiation into myosin heavy chain (MHC)-positive myocytes. These myoctyes begin cell fusion with each other to generate multinucleated myotubes. Typically, most myoblasts become MHC-positive differentiating mononuclear myocytes or myotubes (Figure 2) by day 3-5 in Differentiation Medium.
5. Myoblast Transplantation into Mouse for Skeletal Muscle Regeneration
- Anaesthetize the mouse with Avertin (250 mg/kg) by intraperitoneal (IP) injection.
- Shave the skin hair around on tibialis anterior (TA) muscle. Twenty-four hours before myoblast transplantation, 10 μM CTX (50 μl) is intramuscularly injected into the Nod/Scid mouse TA muscle to induce muscle regeneration via a 31 G insulin syringe through shaved skin (Figure 3).
- Proliferating myoblasts are dissociated with 0.25% Trypsin solution, and centrifuged at 1,000 rpm for 5 min. Aspirate and discard the supernatant. Resuspend 1 x 106 cells with 50 μl of 2% FBS in DMEM. Transfer the suspended cells into a 31 G insulin syringe.
- Recipient mice will be anaesthetized with Avertin (250 mg/kg) by IP injection, and the 1 x 106 myoblasts (Figure 2) are intramuscularly injected into regenerating TA muscle.
- Harvest TA muscle by 1-4 weeks after cell injection for histological analysis (Figure 3).
Freshly isolated quiescent satellite cells display a small, round shape (Figure 1G), and express Pax7 as a definitive marker for quiescent satellite cells. More than 90% of freshly isolated cells express Pax7 (Figure 1H and 1I). Most contaminated cells are from blood cells which do not efficiently grow in vitro following myoblast culture conditions. Thus, satellite cell-derived myoblasts dominate in the culture. Optionally, we can repeat the steps for MS column purification (steps 2.11-2.14) to increase the purity of isolated satellite cells. These quiescent satellite cells will enter the cell cycle within 24 hr after isolation to undergo myogenic precursor cells or myoblasts. These cells can be passaged by trypsinization every 4 days until the cell proliferation rate is reduced. Typically, these cells can be maintained until passage 10. These proliferating myoblasts express MyoD (Figure 2) and Pax7 (data not shown). Normally, more than 99% of cells express MyoD a few days after plating. In the differentiation medium, myoblasts exit the cell cycle, fuse with each other, and become multinucleated myotubes, which express MHC (Figure 2). These ex vivo expanded myoblasts can be utilized for intramuscular cell injection experiments for examination of myoblast contribution to regenerating muscle fibers and self-renewing satellite cells. Twenty-four hours before cell injection, CTX is injected into TA muscles of 2 month-old Nod/Scid immunodeficient mice. Dissociated myoblasts are injected into CTX-induced regenerating muscle (Figure 3). The injected muscle can be harvested a few days to several months after injection. Donor cells are typically genetically labeled with green fluorescent protein (GFP) gene6, β-galactosidase gene16,17, or the alkaline phosphatase gene18 by plasmid transfection, viral vector infection, or preparation from transgenic mice carrying a transgene. Satellite cells from heterozygous Myf5+/nLacZ mice19, in which myogenic cells can be detected after X-gal staining, were isolated. The Myf5+/nLacZ mice carry the nuclear β-galactosidase gene inserted into the Myf5 gene locus, where the β-galactosidase gene expression recapitulates the expression of endogenous Myf5 in both satellite cells and myogenic precursor cells or myoblasts20. Figure 3 shows whole TA muscle staining for the detection of nuclear β-galactosidase-positive donor-derived cells, which include proliferating myoblasts, self-renewing satellite cells, and newly formed muscle fibers. If necessary, these stained TA muscles can be used for histological sections for further immunodetection methods.
Figure 1. Preparation of muscle satellite cells from mouse skeletal muscle. A: Mincing dissected triceps and hind limb muscle by scissors. B: Magnified view of panel A. C: Triturating collagenase-treated minced muscle pieces by 18 G needle. D: Filtrating dissociated muscle preparation by cell strainer. E: MACS separation of dissociated muscle cells by LD column. F: MACS separation of dissociated muscle cells by MS column. G: Freshly isolated satellite cells. H: Pax7-positive freshly isolated satellite cells. I: DAPI staining for panel G.
Figure 2. Culture of isolated satellite cells. A, B, C: Anti-MyoD antibody-positive myoblasts derived from freshly isolated satellite cells. D, E, F: Anti-myosin heavy chain (MHC)-positive differentiating multi-nucleated myotubes.
Figure 3. Intramuscular injection of expanded myoblasts into mouse skeletal muscle. A: X-gal staining of myoblasts isolated from Myf5+/nLacZ mice. B, C: Intramuscular injection of Myf5+/nLacZ myoblasts into TA muscle. D, E: Integration of injected myoblasts isolated from Myf5+/nLacZ mice into regenerating muscle fibers. X-gal staining was performed on TA muscle injected with myoblasts by 1 month.
In this protocol, quiescent satellite cells can be easily purified from adult skeletal muscle of mice by collagenase digestion and surface antibody-mediated MACS separation. This method takes approximately 6 hours and does not need any expensive equipment such as a FACS machine. In addition, this method is relatively inexpensive compared to surface antibody-mediated FACS separation. A higher yield of quiescent satellite cells is also expected in comparison to FACS for this method since FACS laser exposure tends to induce cell death during separation15. Other isolation methods such as preplating or single muscle fiber culturing require a few days to culture, and thus, these methods are good for activated satellite cells or myoblast isolation. However, quiescent satellite cells cannot be purified by these methods. Preplating methods exclude fibroblast contamination on the basis of their adherence difference between activated satellite cells and myoblasts21. Activated satellite cell or myoblast outgrowth occurs during single muscle fiber culture22. We also notice that younger mice (1-2 months old) show a higher yield of quiescent satellite cells (1-5 x 105/mouse) purified by this MACS separation method compared to older mice. However, purity of quiescent satellite cells from damaged muscle is reduced after this surface antibody-mediated MACS purification since the damaged muscle contains more infiltrated blood cells. For the MACS separation of quiescent satellite cells, we utilized CD45, CD31, and Sca-1 as cell surface markers for negative selection. CD45 is a maker for pan-hematopoietic cells; CD31 is a marker for endothelial cells; Sca-1 is a marker for both endothelial cells and interstitial cells23. For the positive selection, we utilized an anti-Integrin α7 monoclonal antibody, which stains quiescent satellite cells as well as some nonmuscle cells in skeletal muscle. Several groups also utilized an anti-Integrin α7 antibody for FACS-based quiescent satellite cell purification, in combination with other positive and negative selection markers7,24. In addition, other groups utilized antibodies against CXCR4 25, CD34 7, Syndecan-3 26, or Syndecan-4 27 as positive selection markers for FACS-based quiescent satellite cell purification. An SM/C-2.6 monoclonal antibody was also used for the positive selection while the epitope for this monoclonal antibody has not been identified yet1. None of these positive selection markers are quiescent satellite cell-specific. Therefore, complete elimination of such nonsatellite cells expressing these positive selection markers is essential for obtaining high purity of quiescent satellite cells after sorting. It might be more useful to use satellite cell-specific cell surface markers such as M-cadherin as a positive selection marker.
Freshly isolated quiescent satellite cells can be used for gene and protein expression profiles, culture experiments to obtain myoblasts and cell transplantation for satellite cell self-renewal experiments, and cell therapies. Previous work demonstrated that gene expression profiles are significantly different from activated satellite cells, which may explain some of the biological differences between these two cell types (such as cells in the dormant phase vs. active cell division)1,28. Recent work has demonstrated that freshly isolated quiescent satellite cells possess significantly higher engraftment and self-renewal activity compared to activated satellite cells or myoblasts when transplanted into regenerating muscle6-7. For example, less than 100 quiescent satellite cells show robust contribution to regenerating muscle fibers as well as to self-renewing satellite cells5,7. Therefore, quiescent satellite cells could be isolated and tested for their potential to efficiently engraft in damaged muscle, their contribution to muscle fiber regeneration, and their improvement of muscle function in DMD patients.
No conflict of interest declared.
We thank Dr. Shahragim Tajbakhsh for providing Myf5+/nLacZ mice. We also thank Alexander Hron and Michael Baumrucker for critical reading of this manuscript. This work was supported by grants from the Muscular Dystrophy Association (MDA) and Gregory Marzolf Jr. MD Center Award.
|Collagenase Type 2||Worthington||CLS-2||100 mg|
|Marigel||BD Biosciences||356234||5 ml|
|Collagen (Rat Tail)||BD Biosciences||354236||100 mg (3-4 mg/ml)|
|Acetic Acid||Sigma-Aldrich||320099-500ML||500 ml|
|bFGF, human, Recombinant||Gibco-Invitrogen||PHG0263||1 mg|
|Bovine Serum Albumin (BSA)||Sigma-Aldrich||A5611-1G||1 g|
|Ham’s F10 Medium||Gibco-Invitrogen||11550-043||500 ml|
|Fetal Bovine Serum (FBS)||Fisher Scientific||3600511||500 ml|
|Horse Serum||Gibco-Invitrogen||26050088||500 ml|
|Phosphate Buffered Saline||Gibco-Invitrogen||14190144||500 ml|
|0.25% Trypsin/EDTA||Gibco-Invitrogen||25200072||500 ml|
|18 G needle with 12 ml Syringe||Fisher Scientific||22-256-563|
|Cell strainer (70 μm)||Fisher Scientific||22-363-548|
|Falcon 50 ml tube||BD Biosciences||352098|
|Falcon 15 ml tube||BD Biosciences||352097|
|10 cm Tissue culture plate||BD Biosciences||353003|
|6 cm Tissue culture plate||BD Biosciences||353004|
|Falcon 10 ml disposable pipette||BD Biosciences||357551|
|Anti-Integrin α7 antibody||MBL International||ABIN487462|
|Anti-PE MicroBeads||Miltenyi Biotec||130-048-801|
|Anti-Mouse IgG MicroBeads||Miltenyi Biotec||130-048-402|
|Mini & MidiMACS Starting Kit||Miltenyi Biotec||130-091-632|
|MS Column||Miltenyi Biotec||130-042-201|
|LD Column||Miltenyi Biotec||130-042-901|
|Cardiotoxin||Sigma Aldrich||C9759-1MG||Stock 10 μM in PBS|
|31 G Insulin syringe||BD Biosciences||328438|
|Refrigerated Microcentrifuge (Microfuge 22R)||Beckman Coulter||368826|
|S241.5 Swinging Bucket Rotor||Beckman Coulter||368882|
|Refrigerated Centrifuge (Allegra X-22R)||Beckman Coulter||392187|
|Nod/Scid immunodeficient mice||Charles River Laboratories||Strain Code 394||Use 2 months old mice|
|10% and 2% FBS DMEM||DMEM (Gibco-Invitrogen #10569010) with 10% or 2% FBS (Fisher Scientific #03600511) and 1% Penicillin/Streptomycin (Gibco-Invitrogen #15640055).|
|0.2% Collagenase solution||Collagenase Type 2 (Worthington, #CLS-2), Stock: 50 ml: 100 mg Collagenase Type 2 in 10% FBS DMEM.|
|10% Matrigel solution||Matrigel (BD Biosciences: #356234) is placed on ice for thawing overnight. Five ml Matrigel is dilute by 45 ml DMEM and 5 ml aliquots are stored at -20 °C until use.|
|Matrigel-coated plate||Five ml of 10% Matrigel solution is placed on ice for thawing and is used for coating 10 cm plate at room temperature for 1 min. The plate is placed in 5% CO2 incubator at 37 °C for 30 min after removing Matrigel solution, and let the plate dry in culture hood for another 30 min. Removed 10% Matrigel solution is stored at -20 °C for reuse.|
|0.01% Collagen solution||Mix to final: 0.01% Collagen (Collagen, Rat Tail: BD Biosciences #354236) in 0.2% acetic acid (320099-500ML) in ddH2O.|
|Collagen-coated plate||Add 5 ml or 2 ml of Collagen solution to a 10 cm or 6 cm tissue culture plate and let sit at room temperature for three hours. Then, aspirate off liquid and allow to dry in culture hood for 30 min to overnight. Plates can be stored at room temperature for several months.|
|bFGF stock solution||bFGF, Human, Recombinant (Gibco-Invitrogen #PHG0263, 1 mg) is dissolved with 0.1% BSA solution consisting of 1 mg BSA (Sigma-Aldrich #A5611-1G) and 2 ml ddH2O (0.5 mg/ml bFGF). Aliquot 20 μl in 500 μl microcentrifuge tubes and kept in -80 °C.|
|Myoblast medium||500 ml HAM’S F10 Medium (Gibco-Invitrogen #11550-043) supplemented with 20% FBS (Fisher Scientific #03600511), Penicillin/streptomycin (Gibco-Invitrogen #15640055), and 10 μg of bFGF (20 μl of bFGF stock).|
|Differentiation medium||500 ml DMEM (Gibco-Invitrogen #10569010) supplemented with 5% Horse serum (Gibco-Invitrogen #26050088) and 1% Penicillin/streptomycin (Gibco-Invitrogen #15640055).|
|10 μM Cardiotoxin stock||1 mg Cardiotoxin (EMD Millipore #217504-1MG) is dissolved with 13.9 ml PBS.|
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