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Summary

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. 

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.

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^1-3. 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 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 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 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 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 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.

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 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. 3. Maintenance 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. 4. Differentiation 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).

Representative Results

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…

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…

Materials

			Materials
Collagenase Type 2	Worthington	CLS-2	100 mg
Marigel	BD Biosciences	356234	5 ml
DMEM	Gibco-Invitrogen	10569010	500 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
Penicillin/Streptmycin	Gibco-Invitrogen	15640055	100 ml
Phosphate Buffered Saline	Gibco-Invitrogen	14190144	500 ml
0.25% Trypsin/EDTA	Gibco-Invitrogen	25200072	500 ml
18G needle with 12cc 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 pipet	BD Biosciences	357551
Anti-CD31 antibody-PE	eBiosciences	12-0311
Anti-CD45 antibody-PE	eBiosciences	30-F11
Anti-Sca1 antibody-PE	eBiosciences	Dec-81
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
31G 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
			Reagents
Name of the reagent			Recipie
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 temprature for 1 minutes. The plate is placed in 5% CO2 incubator at 37°C for 30 minute after removing Matrigel solution, and let the plate dry in culture hood for another 30 minutes. Removed 10% Matrigel solution is stored at -20°C for reusing.
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.