Isolation, culture, and transplantation of muscle satellite cells

J Vis Exp. Author manuscript; available in PMC 2014 Aug 13.

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J Vis Exp. 2014; (86): 10.3791/50846.

Published online 2014 Apr 8. doi:  10.3791/50846

PMCID: PMC4131689

NIHMSID: NIHMS605504

Isolation, culture, and transplantation of muscle satellite cells

Norio Motohashi, Yoko Asakura, and Atsushi Asakura

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Abstract

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.

Keywords: skeletal muscle, muscle stem cell, satellite cell, regeneration, myoblast transplantation, muscular dystrophy, self-renewal, differentiation, myogenesis

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Introduction

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 calcitonin ^1,2,3. Satellite cells have proven to be responsible for muscle regeneration as muscle stem cells. In adult muscle, satellite cells are normally mitotically quiescent ^4-8. Following injury, satellite cells are activated, initiate expression of MyoD, and enter the cell cycle to expand their progeny, termed myogenic precursor cells or myoblasts ³. 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 non-muscle tissues is exploited by myoblast transplantation, a potential therapeutic approach for Duchenne muscular dystrophy (DMD) ⁴, urological dysfunction ⁹, and heart failure ¹⁰. Indeed, myoblasts have been successfully transplanted in the muscle of both mdx (DMD model) mice and DMD patients ^11-14. The injected normal myoblasts fuse with host muscle fibers to improve the histology and function of the diseased muscle. Previous work demonstrated that sub-populations of myoblasts are more stem cell-like and remain in an undifferentiated state longer in muscle during muscle regeneration ⁵. 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 muscle ^5-8. 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) machine ^1,2,6-8. In addition, FACS laser exposure tends to induce cell death during separation, which causes lower yield of quiescent satellite cells ¹⁵. 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.

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Protocol

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/O₂ 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

1.1) Properly sacrifice 1 or 2 young adult mice (3 weeks to 8 weeks).

1.1.1) 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).

1.1.2) 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.

1.2) Wash blood off muscles in PBS and transfer muscles to a new sterile 6 cm plate: 1 plate for 1-2 mice.

1.3) Remove connective tissue, blood vessels, nerve bundles, and adipogenic tissue under a dissection microscope.

1.4) Using scissors for ophthalmology, cut and mince the tissue into a smooth pulp (Fig. 1A, B). Try not to leave large pieces, as they will not be broken down readily by the enzyme solution.

Figure 1

Preparation of muscle satellite cells from mouse skeletal muscle

1.5) 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 minutes.

1.6) Triturate (up and down with an 18G needle) to homogenize mixture (Fig. 1C). Then further incubate the mixture at 37 °C for 15 minutes.

1.7) 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.

1.8) Place a cell strainer (70 μm) onto a Falcon 50 ml tube (Fig. 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.

1.9) Count cell number by hemocytometer. Centrifuge the tubes at 2000 rpm at 4 °C for 5 minutes; aspirate and discard the supernatant.

1.10) Re-suspend with 10 ml of 2% FBS in DMEM. Centrifuge the tubes at 2000 rpm at 4 °C for 5 minutes; aspirate and discard the supernatant.

1.11) Re-suspend with 200 μl of 2% FBS in DMEM and transfer cell suspension into 1.5 ml microcentrifuge tubes. Usually, about 2×10⁶ cells should be harvested from muscles of 1 mouse. Cells will be diluted to a concentration of 1×10⁶ 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.

2.1) 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.

2.2) Wash cells: After incubation, add 1ml of 2% FBS in DMEM into the cell suspension in the 1.5ml tube, and centrifuge at 2000 rpm at 4 °C for 3 minutes. Repeat this step twice.

2.3) Aspirate and discard the supernatant.

2.4) Re-suspend cells with 200 μl of 2% FBS in DMEM, and add 10 μl of Anti-PE Magnetic Beads. Incubate on ice for 30 minutes.

2.5) Wash cells: After incubation, add 1 ml of MACS buffer to the cell suspension in the 1.5ml microcentrifuge tube, and then centrifuge at 2000 rpm at 4 °C for 3 minutes. Repeat this step twice. Note that cells should be washed by MACS buffer before being separated by magnetic column.

2.5.1) Aspirate and discard the supernatant. Re-suspend the cells with 1.0 ml of MACS buffer.

2.6) Set up LD column on a Magnetic board, and rinse the column with 2.0 ml of MACS buffer (Fig. 1E).

2.7) 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.

2.8) Centrifuge at 2000 rpm at 4 °C for 3 minutes, aspirate and discard the supernatant.

2.9) Re-suspend cells with 200 μl of 2% FBS in DMEM, and add 10 μl of Anti-Mouse IgG Magnetic Beads. Incubate on ice for 30 minutes.

2.10) Wash cells: After incubation, add 1 ml of MACS buffer into the cell suspension in 1.5ml tube, and then centrifuge at 2000 rpm at 4 °C for 3 minutes. Aspirate and discard the supernatant. Repeat this step twice. Re-suspend cells with 500 μl of MACS buffer.

2.11) Set up MS column on a Magnetic board, and rinse the column with 500 μl of MACS buffer (Fig. 1F).

2.12) Transfer suspended cell solution onto the MS column, and discard the flow-through fraction (Integrin α7-negative cells).

2.13) Rinse with 1ml of MACS buffer, and repeat this step twice.

2.14) 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.

2.15) Centrifuge at 2000 rpm at 4 °C for 3 minutes, aspirate and discard the supernatant.

2.16) Re-suspend 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) (Fig. 1G). Note that 1 to 2 × 10⁵ cells can potentially be isolated from 1 intact mouse muscle.

3) Maintenance

3.1) Feed cells every other day with Myoblast medium. The appearance of growing myoblasts is of a small and round shape expressing MyoD (Fig. 2) and Pax7 (data not shown) in their nucleus.

Figure 2

Culture of isolated satellite cells

3.2) 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 minutes in a CO₂ incubator and collect dissociated cells with Myoblast medium. After centrifuge cells (1000 rpm for 5 minutes), suspend with Myoblast medium and re-plate 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.

4) Differentiation

4.1) Re-feed with Differentiation Medium every other day.

4.2) 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 (Fig. 2) by day 3 to 5 in Differentiation Medium.

5) Myoblast transplantation into mouse for skeletal muscle regeneration

5.1) Anaesthetize the mouse with Avertin (250 mg/kg) by intraperitoneal (IP) injection.

5.2) 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 31G insulin syringe through shaved skin (Fig. 3).

Figure 3

Intramuscular injection of expanded myoblasts into mouse skeletal muscle

5.3) Proliferating myoblasts are dissociated with 0.25% Trypsin solution, and centrifuged at 1000 rpm for 5 minutes. Aspirate and discard the supernatant. Resuspend 1×10⁶ cells with 50 μl of 2% FBS in DMEM. Transfer the suspended cells into a 31G insulin syringe.

5.4) Recipient mice will be anaesthetized with Avertin (250 mg/kg) by IP injection, and the 1×10⁶ myoblasts (Fig. 2) are intramuscularly injected into regenerating TA muscle.

5.5) Harvest TA muscle by 1-4 weeks after cell injection for histological analysis (Fig. 3).

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Representative Results

Freshly isolated quiescent satellite cells display a small, round shape (Fig. 1G), and express Pax7 as a definitive marker for quiescent satellite cells. More than 90% of freshly isolated cells express Pax7 (Fig. 1H, I). 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 (2.11-2.14) to increase the purity of isolated satellite cells. These quiescent satellite cells will enter the cell cycle within 24 hours 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 (Fig. 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 (Fig. 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 (Fig. 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) gene ⁶, β-galactosidase gene ^16,17, or the alkaline phosphatase gene ¹⁸ by plasmid transfection, viral vector infection, or preparation from transgenic mice carrying a transgene. Satellite cells from heterozygous Myf5^+/nLacZ mice ¹⁹, 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 myoblasts ²⁰. Fig. 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.

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Discussion

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 separation ¹⁵. Other isolation methods such as pre-plating 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. Pre-plating methods exclude fibroblast contamination on the basis of their adherence difference between activated satellite cells and myoblasts ²¹. Activated satellite cell or myoblast outgrowth occurs during single muscle fiber culture ²². We also notice that younger mice (1-2 months old) show a higher yield of quiescent satellite cells (1-5 × 10⁵ per 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 cells ²³. For the positive selection, we utilized an anti-Integrin α7 monoclonal antibody which stains quiescent satellite cells as well as some non-muscle 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 markers ^7,24. In addition, other groups utilized antibodies against CXCR4 ²⁵, CD34 ⁷, Syndecan-3²⁶, 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 yet ¹. None of these positive selection markers are quiescent satellite cell-specific. Therefore, complete elimination of such non-satellite 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 muscle ^6-7. For example, less than 100 quiescent satellite cells show robust contribution to regenerating muscle fibers as well as to self-renewing satellite cells ^5,7. 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.

Table 1

Materials.

Table 2

Reagents.

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Acknowledgments

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.

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Footnotes

Disclosure No conflict of interest declared.

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Contributor Information

Norio Motohashi, Stem Cell Institute, Paul and Sheila Wellstone Muscular Dystrophy Center, Department of Neurology University of Minnesota Medical School ; Email: ude.nmu@sahotomn.

Yoko Asakura, Stem Cell Institute, Paul and Sheila Wellstone Muscular Dystrophy Center, Department of Neurology University of Minnesota Medical School ; Email: ude.nmu@arukasay.

Atsushi Asakura, Stem Cell Institute, Paul and Sheila Wellstone Muscular Dystrophy Center, Department of Neurology University of Minnesota Medical School.

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