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Stem Cells 2004;22:1305-1320 www.StemCells.com
© 2004 AlphaMed Press

Cellular and Molecular Regulation of Skeletal Muscle Side Population Cells

Annette P. Meesona, Thomas J. Hawkea, Sarabeth Grahama, Nan Jianga, Joel Eltermana, Kelley Hutchesonb, J. Michael DiMaiob, Teresa D. Gallardoa, Daniel J. Garrya,c,d

a Departments of Internal Medicine,
b Cardiothoracic Surgery,
c Molecular Biology, and
d The Donald W. Reynolds Cardiovascular Research Center, University of Texas Southwestern Medical Center, Dallas, Texas, USA

Key Words. Side population cells • Myoblast • Foxk1 • Skeletal muscle • Bone marrow

Correspondence: Daniel J. Garry, M.D., Ph.D., NB11.118A, 5323 Harry Hines Boulevard, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8573, USA. Telephone: 214-648-1654; Fax:214-648-1450; e-mail: daniel.garry{at}utsouthwestern.edu


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Muscle progenitor cells (satellite cells) function in the maintenance and repair of adult skeletal muscle. Side population (SP) cells are enriched in repopulating activity and also reside in adult skeletal muscle. In this study, we observed that Abcg2 is a determinant of the SP cell phenotype. Using reverse transcription polymerase chain reaction and immunohistochemical techniques, we localized Abcg2-expressing cells in the interstitium and in close approximation to the vasculature of adult skeletal muscle. Muscle SP cells are able to differentiate into myotubes and increase in number after cardiotoxin-induced muscle injury. Similar to myogenic progenitor cells, muscle SP cells express Foxk1 and are decreased in number in Foxk1 mutant skeletal muscle. Using emerging technologies, we examine the molecular signature of muscle SP cells from normal, injured, and Foxk1 mutant skeletal muscle to define common and distinct molecular programs. We propose that muscle SP cells are progenitor cells that participate in repair and regeneration of adult skeletal muscle.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Adult skeletal muscle is capable of self-repair in response to extreme training, injury, or myopathic diseases such as muscular dystrophy [1, 2]. The ability for self-repair or regeneration is attributable to a rare population of myogenic progenitor cells (MPCs), also referred to as satellite cells [3]. The MPCs are capable of self-renewal and are arrested at an early stage of the myogenic program such that they do not express any of the myogenic basic helix-loop-helix proteins of the MyoD family [2, 4]. In response to an injury, the quiescent MPCs are activated, proliferate, and withdraw from the cell cycle to form differentiated myotubes, and ultimately restore the skeletal muscle architecture.

Foxk1 is a member of the forkhead/winged helix family of transcription factors and is expressed in the MPC population, where it functions as a regulator of the cell cycle [5, 6]. Mice lacking Foxk1 have a growth deficit and severely impaired muscle regeneration [7]. This decrease in regenerative capacity is attributable, in part, to a decrease in MPC number, impaired MPC activation, and impaired MPC proliferation in Foxk1-deficient skeletal muscle [6]. Studies undertaken in mice lacking both Foxk1 and p21 suggest that p21 is a downstream target for Foxk1, but other target genes for this forkhead/winged helix transcription factor are unknown [6].

Recently, an additional stem cell population referred to as side population (SP) cells has been identified in most adult tissues, including skeletal muscle [8, 9]. Using flow cytometry, these SP cells are isolated from adult tissues based on their ability to efflux Hoechst 33342 dye [10, 11]. This ability to efflux Hoechst dye is attributable to the ATP binding cassette (ABC) half-transporter, Abcg2, which has been shown to be the molecular determinant of the SP cell phenotype [12, 13]. SP cells have stem cell properties, because limited numbers of SP cells isolated from adult bone marrow were able to reconstitute the irradiated mdx (dystrophin mutant) mouse bone marrow. Later these cells were recruited from the bone marrow to contribute to differentiated myotubes during muscle regeneration [14]. Previous studies suggest that SP cells may be the precursors of the MPC (satellite) population and require Pax7 for the specification of the satellite cell, because Pax7-deficient mice lack satellite cells but contain a complete repertoire of SP cells [15]. Alternatively, SP cells may represent a second distinct progenitor or stem cell population that is resident in adult skeletal muscle. Although these elegant studies provide additional support for the role of skeletal muscle SP (SMSP) cells as a progenitor/stem cell population, the molecular regulation of this cell population is illdefined.

In the present study, we use morphological, molecular, and transcriptome analyses to enhance our understanding of the SP cell population isolated from wild-type (WT), injured, and Foxk1 mutant adult skeletal muscle. Comparison of these SP cell populations with embryonic stem cells and WT myoblasts additionally defines common and distinct molecular signatures for the respective SP cell populations. Collectively, this strategy will enhance our understanding of the SP cell population and impact therapeutic applications in the treatment of myopathic diseases such as muscular dystrophy.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Transgenic Mice
Two- to 6-month-old C57Bl/6 WT, C57Bl/6 Tie2–green fluorescent protein (GFP), and C57Bl/6 Foxk1-null mice were maintained and genotyped as previously described [6, 16]. Adult ROSA26 mice were purchased from Jackson Laboratories (Bar Harbor, ME), and all mice were maintained according to the guidelines of the NIH and the Institutional Animal Care and Use Committee at University of Texas Southwestern Medical Center.

Cardiotoxin-Induced Muscle Injury
Cardiotoxin (150 µl of 10 µM; Calbiochem, La Jolla, CA) was delivered intramuscularly to the hindlimbs of adult mice as previously described [5]. At specified intervals after injury, mice were euthanized and hindlimb skeletal muscles were removed and processed for morphological or fluorescence-activated cell sort (FACS) analyses. In vivo differentiation assays of SMSP cells were performed in cardiotoxin-injured skeletal muscle of adult severe combined immunodeficiency (SCID) male mice. Briefly, using flow cytometry, SMSP cells were isolated from the ROSA26 adult mice (which constitutively express lacZ) and 250 SP cells (in 15 µl phosphate-buffered saline [PBS]) were delivered intramuscularly into the injured/regenerating gastrocnemius muscle of SCID mice. Ten days later, the muscles were harvested, embedded, sectioned using a Leica CM 3000 cryostat, and examined for LacZ expression as previously described [5].

Isolation of SP Cells Using FACS
Hindlimb skeletal muscles were harvested from adult mice, and bone marrow was extracted from the femurs and tibias as previously described [17]. Briefly, the skeletal muscle was digested with pronase (10 mg/ml) and cells were separated using a percoll gradient (40%/70%) and resuspended at 1.0 x 106 cells/ml in Hank’s buffer containing 2% fetal calf serum (FCS). Bone marrow was prepared as previously described [10, 11]. The respective cell populations were stained by the addition of Hoechst 33342 (bone marrow, 5 µg/ml; skeletal muscle, 12.5 µg/ml) for 90 minutes at 37°C in the presence and absence of verapamil (100 µM) and fumitremorgin C (FTC; 10 µM). The verapamil or FTC was added immediately before the addition of the Hoechst dye. Cell populations were then rinsed with PBS, pelleted, and resuspended in 1 ml of Hank’s media and maintained at 4°C before FACS analysis (MoFlo, Cytomation, Inc., Fort Collins, CO). After Hoechst staining, in selected experiments, immunostaining was performed using a FITC (fluorescein isothiocyanate) c-Kit fluorophore-conjugated antiserum (Becton, Dickinson, Palo Alto, CA) at a concentration of 1 µg/ml for 20 minutes. Cells for microarray analysis were sorted into Tripure and stored at –80°C. FACS profiles were recorded using Cytomation Summit software and transferred into PhotoShop 5.5 files. Student’s t-tests were performed to test for significant differences. Data are presented as mean ± standard error of the mean unless otherwise noted.

Immunohistochemistry
Hindlimb skeletal muscle was immersion fixed in 4% paraformaldehyde (pH 7.2) overnight at 4°C, rinsed in PBS, and paraffin embedded. Four-micron-thick sections were deparaffinized in xylene and rehydrated to PBS. Sections were permeabilized in 0.3% Triton X-100 in PBS for 5 minutes for intracellular antigens. All sections were blocked with the respective 5% normal serum for 30 minutes. Nonspecific binding to endogenous peroxidases was blocked by incubating sections in 0.6% hydrogen peroxide/methanol. Sections were then incubated with the primary antisera in a humid chamber at 4°C overnight [18]. Primary antisera used in this study included a polyclonal rabbit anti-ABCG2 serum (1:1,600 dilution, kindly provided by Dr. Susan Bates, National Cancer Institute) or a polyclonal rabbit anti-c-Kit serum (0.5 µg/ml; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The next day, the sections were rinsed with PBS and incubated with the respective secondary antiserum for 30 minutes at room temperature. The secondary antisera included either the biotinylated goat anti-rabbit serum (1:200 dilution;Vector Laboratories, Burlingame, CA), which was detected with FITC-conjugated streptavidin (1:50 dilution; Vector Laboratories) or with biotinylated goat anti-rabbit serum (1:200 dilution; Vector Laboratories) followed by horseradish peroxidase streptavidin (1:500 dilution; Vector Laboratories) that was visualized with dimethylaminoazobenzene (Dako, Carpinteria, CA). Images were recorded using a Leitz Laborlux S microscope (Bannockburn, IL) and Scion imaging software (Scion, Frederick, MD).

Using FACS, SP cells were isolated and cyto-spun onto plus-coated slides, air dried, and fixed in 4% paraformaldehyde for 10 minutes at 4°C. The cells were rinsed in PBS, permeabilized in 0.3% triton in PBS, and incubated with an affinity-purified rabbit anti-Foxk1 serum (1:100 dilution) overnight at 4°C in a humid chamber [5]. The following day, the cells were rinsed with PBS, incubated with FITC-conjugated goat anti-rabbit serum (1:50 dilution; Jackson Immunoresearch, West Grove, PA) for 30 minutes at room temperature, rinsed with PBS, and mounted with Vectashield (Vector Laboratories). Final images were generated using Adobe PhotoShop 5.5 software package (Adobe, San Jose, CA).

Cell Culture
After FACS of SMSP cells and MPCs (n = 3 for both cell populations) into 24-well Matrigel-coated plates, cells were cultured in MPC differentiation media, which included Dulbecco’s modified Eagle’s medium containing 2% normal horse serum, 0.5% penicillin/streptomycin, HEPES buffer (50 mM), transferrin (10 µg/ml), and insulin (10 µg/ml), and observed daily for differentiation into myotubes. At specified periods, cells were rinsed with PBS, fixed with 4% paraformaldehyde for 5 minutes at 4°C, and stained with Hoechst 33342 (1 mg/ml, diluted 1:1,000) for 10 minutes. The cells were rinsed and imaged using an Olympus IM2 inverted microscope and Scion imaging software.

RNA Isolation and Reverse Transcription Polymerase Chain Reaction
Total RNA was isolated from adult SMSP and MP, adult aorta, adult femoral artery, neonatal myoblasts, neonatal pupillary membrane, and hyaloid vasculature using the Tripure Isolation Kit (Roche, Indianapolis). Semiquantitative reverse transcription polymerase chain reaction (RT-PCR) analysis was performed as previously described using primers that span an intron [18]. PCR primer pairs used for this study included the following: Abcg2 forward: 5'-GTGGCATC TCTGGAGGAGAA-3'; reverse: 5'-TCCTGAGCTCCTG GAAGTTG-3'; Foxk1 forward: 5'-TACTTCATCAAAG TCCCTCGGTC-3'; reverse: 5'-GTACTCTGGAACAGAG GCTAACTT-3'; PECAM (CD31) forward: 5'-GACACTA CACCTGCAAAGTG-3'; reverse: 5'-GCACCGAAGTA CCATTTCAC-3'; Ly6a forward: 5'-CTCTGAGGATGGAC ACTTCT-3'; reverse: 5'-GGTCTGCAGGAGGACTGAGC -3'; c-Kit forward: 5'-CCAGGAATATCCTCCTCACTCA -3'; reverse: 5'-CCAAGTAACCATCACAGAA-3'; angio-poietin2 (Agpt2) forward: 5'-CACAGCGAGCAGCTACA GTC-3'; reverse: 5'-GGATAGCAACCGAGCTCTTG-3'; Desmin forward: 5'-GTGAAGATGGCCTTGGATGT-3'; reverse: 5'-CTTCAGGAGGCAGTGAGGAC-3'; GAPD forward: 5'-GTGGCAAAGTGGAGATTGTTGCC-3'; reverse: 5'-GAT GATGACCCGTTTGGCCTCC-3'.

Probe Labeling and Affymetrix Array Hybridization
Total RNA was isolated from the neonatal skeletal muscle myoblasts and FACS-sorted SP and MP cells. Oligonucleotide array hybridizations were carried out according to the Affymetrix protocol as described previously [19]. Total RNA was isolated from the respective cell samples (2,000 to 4,000 cells) using the Tripure Isolation Kit, amplified using a modified T7 transcription reaction (two rounds of amplification), to produce cDNA. The cDNA was converted to biotin-labeled cRNA by using the Enzo BioArray high-yield RNA transcript labeling kit (Enzo Biochem, New York), fragmented, hybridized to the high-density oligonucleotide Murine Genome Array U74Av2 GeneChip (Affymetrix, Santa Clara, CA) for 16 hours, and scanned according to the manufacturer’s protocol (sorts and microarray hybridizations were performed in duplicate). Microarray Suite (MAS) Version 5.0 Software (Affymetrix) was used for normalization, scaling of chip intensities, calculating presence and absence calls, and fold-change. A 95% confidence bound was used for all calculations.

Ultrastructural Analysis
FACS-sorted SMSP cells were fixed in 3% glutaraldehyde/PBS, pelleted by centrifugation at 3,000 rpm for 3 minutes, washed in PBS, and then post-fixed in 1% buffered osmium tetroxide for 30 minutes. The fixed cells were dehydrated in ethanol, embedded in Spurr resin, and polymerized overnight at 60°C. Semi-thin sections were stained with Toludine Blue, and ultra-thin sections were stained with uranyl acetate and lead citrate. Ultra-thin sections were examined using a JEOL 1200 EXII Transmission Electron Microscope.

Online Supplementary Material
Supplementary material provided includes all microarray data used in this study and duplicate arrays of all cell populations examined using cells collected on separate occasions.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Abcg2 Is a Determinant of the SP Cell Phenotype
Using dual-wavelength flow cytometry, SP cells were isolated from several adult tissues based on the ability of the cell population to efflux Hoechst 33342 dye [10, 11]. Recent studies have demonstrated that the ability of SP cells to efflux Hoechst 33342 dye is dependent on the ABC half-transporter Abcg2 [12, 20, 21]. Furthermore, the fungicide FTC has been reported to be a specific inhibitor of Abcg2, where it functions to inhibit the Abcg2-associated ATPase activity [22]. Using these protocols, we isolated SP cells from adult murine skeletal muscle (Fig. 1AGo) and bone marrow (Fig. 1CGo). Routinely, the absolute number of SP cells obtained from bone marrow exceeded that obtained from adult skeletal muscle. Furthermore, the ability of these SP cell populations to efflux Hoechst dye was inhibited equally well by either the calcium channel blocker verapamil (data not shown) or after the addition of FTC (Figs. 1B, 1DGo). Preliminary studies undertaken in our laboratory reveal that overexpression of Abcg2 in C2C12 myoblasts confers the SP cell phenotype and that the ability to efflux the dye in the Abcg2-overex-pressing C2C12 myoblasts can be inhibited completely by FTC (C. Martin, D. Garry, personal observations).



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  		Figure 1. Isolation of SP cell populations using flow cytometry. SP cell populations were isolated from adult skeletal muscle and adult bone marrow, respectively (A, C) using dual-wavelength fluorescence-activated cell sorter and Hoechst 33342 dye. Note that SP cells are located within the gated regions. Addition of fumitremorgin C to skeletal muscle (B) and bone marrow (D) cell preparations inhibited the ability of the SP cells to efflux the Hoechst 33342 dye. Abbreviations: MP, main population; SP, side population.

 
Morphological Characteristics and Differentiation Capacity of SMSP
SP cells were isolated from noninjured WT adult mouse hind-limb skeletal muscle using Hoechst 33342 dye efflux and FACS analysis. Sorted cells were stained with the Hema3 kit (Fischer Diagnostics, Middleton, VA) and examined at the light microscopic level. We observed that the SMSP cells were relatively small (6.6 ± 0.1 µm, n = 154) and had a high nuclear-to-cytoplasmic ratio (Fig. 2AGo). Ultrastructural examination of SMSP cells additionally confirmed that these small mononucleated cells had a high nuclear-to-cytoplasmic ratio, with few intracellular organelles (Fig. 2BGo). A high nuclear-to-cytoplasmic ratio is characteristic of quiescent stem cell populations, such as embryonic stem cells, and somatic stem cell populations, such as the MPCs (i.e., satellite cells) in adult skeletal muscle [2]. SMSP are uniformly smaller (Fig. 2CGo) than the embryonic stem (ES) cells (Fig. 2DGo) that are propagated in culture.



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  		Figure 2. Morphological analysis of SMSP cells. Microscopic analysis of wild-type SMSP cells using light microscopy (A) and electron microscopy (B) revealed a high nuclear-to-cytoplasmic ratio in SMSP cells. Comparison of SMSP cells (C) with ES cells (D) revealed morphological differences (i.e., size) between these two cell populations. Arrowheads denote the SMSP cells (C) and the ES cells (D). Side population and myogenic progenitor cells were sorted and cultured to evaluate their differentiation capacity. SMSP cells did not differentiate (E), whereas SMMP cells attached to the culture dish and differentiated after serum removal (F). In contrast, sorted SMSP (G) and SMMP (H) readily form multinucleated (stained with Hoechst dye) myotubes when cultured with myogenic progenitor differentiation media and on Matrigel-treated plates. SMSP cells are capable of differentiating into myotubes in vivo. Two days after cardiotoxin-induced skeletal muscle injury, 250 SMSP cells isolated from adult ROSA26 skeletal muscle were intramuscularly delivered into the injured skeletal muscle. Ten days later, the skeletal muscle was harvested and stained with X-gal. Note ß-galactosidase–positive fibers (blue) present within the regenerating skeletal muscle (I). Sorted SMSP cells were cytospun onto slides and were observed to express Foxk1 (arrowheads) using immunofluorescence techniques (J). Scale bars = 20 µm (A), 500 nm (B), and 20 µm (C–J). Abbreviations: ES, embryonic stem; SMMP, side population main population; SMSP, skeletal muscle side population.

 
Previous studies observed that isolated SMSP cells differentiate slowly (2 weeks compared with 1 week for differentiation of skeletal muscle MP [SMMP]) into a mixture of myoblasts and fibroblasts [14] or that isolated SMSP cells cultured in myoblast growth medium did not give rise to myogenic progenitors unless cultured in the presence of primary myoblasts [23]. In the present study, we observed that sorted SMSP cells are viable but relatively quiescent, without any evidence of differentiation when cultured in F10 media supplemented with 20% FCS (Fig. 2EGo). In contrast, SMMP cells readily attach to the culture dish and differentiate even under high serum culture conditions (Fig. 2FGo). No cells exhibiting the morphology of the MP cells were detected in the isolated SP cell cultures. We further observed that sorted SMSP (Fig. 2GGo) and MP (Fig. 2HGo) cells cultured individually on Matrigel-pretreated plates in the presence of a MPC-differentiation media formed differentiated multinucleated myotubes but at different rates (3 days for SMMP compared with 7 days for SMSP; n = 3 separate experiments). It is possible that the Matrigel provides a surface similar to the extracellular matrix in skeletal muscle, allowing the SP cells to adhere and differentiate when exposed to this matrix [24].

Having established that the SMSP cells were capable of differentiation in vitro, we examined their ability to repopulate injured skeletal muscle. Two days after cardiotoxin-induced skeletal muscle injury, 250 SP cells (isolated from the skeletal muscle of the ROSA26 mouse model in which all cells constitutively express ß-galactosidase) were delivered intramuscularly into the regenerating skeletal muscle. After skeletal muscle regeneration (10 days after delivery of the cells), we observed evidence of ß-galactosidase–positive fibers (Fig. 2IGo). These results support the conclusion that limited SP cell numbers are capable of competing with endogenous progenitor cell populations in regenerating skeletal muscle to promote muscle repair. These results additionally establish that the SMSPs are capable of differentiation into skeletal muscle after the exposure to in vitro and in vivo environments.

We had previously established that Foxk1 was expressed in MPCs (satellite cells) using light microscopic and ultra-structural immunohistochemical techniques [5]. Although the relationship between SMSP and MPC is ill defined, we undertook immunohistochemical techniques to examine Foxk1 expression in sorted SMSP cells. We observed that a subpopulation of the SMSP cells expressed Foxk1 (Fig. 2JGo). These results establish that Foxk1 is expressed in skeletal muscle progenitor cells (satellite or MPC and SP cells) [5, 7].

Abcg2-Expressing Cells Are Present in Adult Skeletal Muscle
In addition to our results, previous studies support the conclusion that Abcg2 is a determinant of the SP cell phenotype [12]. Using immunohistochemical techniques, we observed rare Abcg2-expressing cells in uninjured adult skeletal muscle (data not shown). After cardiotoxin-induced skeletal muscle injury (5 days after injury), we observed an increase in Abcg2-positive cells, which were in close approximation to vascular structures (Fig. 3AGo). To further corroborate our immunohistochemical results, we used RT-PCR techniques and primers spanning an intron and observed Abcg2 expression in the SP cells of skeletal muscle, the capillaries of the postnatal pupillary membrane and hyaloid vasculature, and the aorta (Fig. 3BGo). Abcg2 expression was absent in the MP population and isolated myofibers (n = 400 myofibers; performed in triplicate). Foxk1 was expressed in all of the samples, consistent with its expression in SP cells and MPCs. Furthermore, Ly6a (Sca-1), c-Kit, and Agpt2 are all expressed in SMSP cells, whereas the panendothelial marker PCAM (CD31) and the intermediate filament protein desmin (which is expressed in differentiated muscle) were absent in SMSP cells (Fig. 3BGo).



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  		Figure 3. Abcg2 is expressed in SP cells. (A): Using immunohistochemical techniques, Abcg2-expressing cells (arrowheads) are located within injured wild-type muscle and closely associated with the vasculature (*). (B): RT polymerase chain reaction analysis reveals Abcg2 expression in the SP cell population isolated from skeletal muscle, the capillaries of the postnatal pupillary membrane (Cp), and the adult aorta (Ao). Note the absence of Abcg2 transcripts in SP myogenic progenitor and in isolated muscle fibers (SF). Foxk1 is expressed in skeletal muscle SP and MP cell populations, myofibers, capillaries of the postnatal pupillary membrane, and aorta, consistent with the expression of Foxk1 in SP and MP cell populations. Ly6a (Sca-1), cKit, and Agpt2 are all expressed in the SP cell populations, whereas the intermediate filament protein, desmin, is not expressed in SP cells but is expressed in differentiated muscle cells associated with isolated fibers and vascular structures (Cp and Ao). Pecam (CD31) is expressed in the Cp and Ao specimens. Gapd expression was used as a loading control. (C): Using Hoechst 33342 dye and dual-wavelength fluorescence-activated cell sorter analysis of the Tie2-GFP transgenic mouse heart, 84% (average of two preparations; 82% and 86%) of SMSP cells were GFP+. (D): This percentage was confirmed when the sorted skeletal muscle SP cells were examined using fluorescent microscopy (arrowheads mark GFP-expressing SP cells). Scale bar = 20 µm for (A) and 50 µm for (D). Abbreviations: MP, main population; RT, reverse transcriptase; SP, side population.

 
The angiopoietins are growth factors that bind and activate Tie2/Tek, which is a receptor tyrosine kinase. Tie2 signal transduction pathways are involved in cell survival and cell migration [25]. To additionally examine the Abcg2 expression in vascular structures, we used the Tie2-GFP transgenic mouse model [16] to determine the relationship between the SP cells and progenitor cell populations, because Tie2 is expressed in selected progenitor cell populations (hematopoietic progenitors) and endothelial cells throughout development and in the adult [25, 26]. Using FACS analysis, we observed that most (84%) of the sorted SMSP cells expressed GFP (Fig. 3CGo). These results were additionally confirmed using epifluorescent microscopy, because most of the sorted SP cells expressed GFP (Fig. 3DGo). In addition, these results are additionally supported by the expression of angiopoietin2 in the SMSP population (Fig. 3BGo). We conclude from these studies that most of the SMSP cells share signaling pathways with endothelial/hematopoietic precursor cell populations.

Decreased SP Cell Numbers in Foxk1-Null Skeletal Muscle That Increase after Muscle Injury
We had previously shown that mice lacking Foxk1 have a severe impairment in muscle regeneration attributable, in part, to decreased MPC numbers [6]. Therefore, we examined the SMSP population in Foxk1-deficient skeletal muscle. Using flow cytometry, we have reproducibly isolated SMSP cells from WT skeletal muscle (Fig. 4AGo). Routinely, we obtain approximately 2,000 to 4,000 SP cells from the hindlimbs of eight adult C57Bl/6 mice. The ability of the SP cells to efflux Hoechst dye is inhibited with the fungicide FTC, which is an Abcg2 inhibitor [22] (Fig. 4BGo). We further observed that Foxk1 adult mice had approximately half the number of SMSP (Fig. 4CGo) compared with the age- and gender-matched WT SMSP (Fig. 4AGo). After cardiotoxin-induced skeletal muscle injury in WT adult mice, we observed more than a 4.5-fold increase in SP cells 5 days after the injury (0.15 ± 0.01% at baseline, which increases to 0.72 ± 0.04%, n = 3; p < .05), consistent with the hypothesis that this cell population participates in muscle repair (Figs. 4D, 4FGo). Ten days after injury, the WT muscle architecture is largely restored and the SMSP approaches baseline levels (Fig. 4FGo). Foxk1 mutant skeletal muscle has decreased numbers of SMSP in unperturbed skeletal muscle, and this cell population increases after skeletal muscle injury (0.11 ± 0.01% at baseline, which increases to 0.39 ± 0.04%, n = 3; p < .05 at 5 days after injury). At all time periods after cardiotoxin-induced muscle injury, Foxk1 mutant skeletal muscle had fewer numbers of SMSP compared with WT controls (Fig. 4FGo). These results support the conclusion that although Foxk1 SMSP cells are decreased in number, they respond similarly to the signals and cues as WT control SP cells after muscle injury. These results may additionally explain the impaired regenerative capacity of the Foxk1 mutant skeletal muscle.



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  		Figure 4. SP cells increase after injury but are decreased in Foxk1 skeletal muscle. (A): Representative FACS profile of skeletal muscle SP cells. Note the SP cells are located in the gated region and account for 0.21% of the total cell population. (B): Inhibition of the SP cell phenotype after the addition of the Abcg2 inhibitor, fumitremorgin C. (C): Fluorescence-activated cell sorter profile reveals fewer SP cells in the Foxk1 mutant muscle compared with WT skeletal muscle. (D): Increased SP cell numbers (compared with uninjured skeletal muscle in A) are observed 5 days after of WT skeletal muscle. (E): Increased SP cell numbers (compared with uninjured Foxk1 skeletal muscle) 5 days after cardiotoxin injury in Foxk1-null skeletal muscle. Note that the increase in SP cell numbers in injured Foxk1-null skeletal muscle is less than injured WT skeletal muscle. (F): Quantitation of the SP cell numbers in WT and Foxk1-injured skeletal muscle. Note that at each time period, WT skeletal muscle has increased numbers of SP cells (n = 3 at each time period; *p < .05). Data are presented as mean ± standard error of the mean. Abbreviations: MP, myogenic progenitor; SP, side population; WT, wild-type.

 
Foxk1–/–Adult Mice Have an Impaired Muscle Regenerative Capacity
Foxk1 mutant mice have severely impaired muscle regeneration because of decreased numbers of MPCs and SP cells. Ten days after cardiotoxin-induced muscle injury, WT skeletal muscle architecture is largely restored (Fig. 5AGo). In contrast, 10 days after injury, Foxk1 skeletal muscle is characterized by persistent myonecrosis and a hypercellular response with rare evidence of newly regenerated myofibers (Fig. 5BGo). We additionally evaluated the contribution of SP cells, which expressed the stem cell marker c-Kit (the receptor for stem cell factor) in unperturbed and injured WT and Foxk1-null skeletal mice. Using flow cytometry in combination with a FITC-conjugated c-Kit antibody, the SMSP from unperturbed WT and Foxk1 skeletal muscle expresses low levels of c-Kit, which is in agreement with previous observations. Five days after injury, c-Kit expression in the SMSP population from WT skeletal muscle increased more than eightfold (1.7 ± 0.4% c-Kit+ SP cells at baseline, increasing to 14.6 ± 0.8%, n = 3; p < .005; Fig. 5CGo) and approached baseline levels by 10 days after injury (2.0 ± 0.6%, n = 5). In contrast, the c-Kit expression in Foxk1-null SMSP cells was markedly delayed and did not increase until 10 days after injury (0.7 ± 0.04% c-Kit+ SP cells at baseline, increasing to 8.9 ± 2.1%, n = 3; p < .05; Fig. 5CGo). These results were further confirmed using immunohistochemical techniques for c-Kit expression in unperturbed and injured skeletal muscle. Low levels of c-Kit expression were observed in uninjured WT and Foxk1-null skeletal muscle (data not shown). After muscle injury, c-Kit–expressing cells increased abundantly by 5 days (Fig. 5DGo) and were largely absent by 10 days after injury in WT skeletal muscle (Fig. 5FGo). The temporal expression pattern for c-Kit expression differed in Foxk1-injured skeletal muscle, because c-Kit+ cells were absent at 5 days (Figs. 5EGo) but increased by 10 days after injury (Fig. 5GGo). At all time periods during the repair process, the c-Kit–expressing cells were less in Foxk1 skeletal muscle compared with WT regenerating skeletal muscle.



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  		Figure 5. Morphological assessment of the skeletal muscle of WT and Foxk1-null mice after cardiotoxin injury. Hematoxylin and eosin–stained tissue sections of cardiotoxin-injured WT and Foxk1-null skeletal muscle. (A): Ten days after injury, WT skeletal muscle has many centronucleated myofibers (arrowheads) corresponding to complete regeneration. (B): In contrast, Foxk1-null skeletal muscle has severely impaired regeneration characterized by a hypercellular myonecrotic state with only rare centronucleated myofibers 10 days after injury. (C): Percentage of WT and Foxk1–/– skeletal muscle SP cells (days 0, 5, and 10 after injury) that are c-Kit+. Note an eightfold increase in c-Kit+ SP cells in the WT 5-day injured skeletal muscle (1.7 ± 0.4% c-Kit+ SP cells at baseline, increasing to 14.6 ± 0.8%, n = 3; *p < .005), whereas there is a significant delay (c-Kit+ cells peak at 10 days after injury to 8.9 ± 2.13%; n = 3 for each sample; *p < .05) in the Foxk1-injured skeletal muscle. (D): Immunohisto-chemical expression of c-Kit+ cells (arrowheads) in regenerating skeletal muscle reveals increased c-Kit expression 5 days after injury in WT skeletal muscle with a relative absence of cells present in Foxk1-null skeletal muscle (E). (F): c-Kit–expressing cells are absent in the 10-day regenerated WT skeletal muscle, whereas a small population of c-Kit+ cells (arrowheads) are observed in the Foxk1-null skeletal muscle 10 days after injury (G). Scale bars = 20 µm (A, B, D–G). Abbreviations: SP, side population; WT, wild-type.

 
SP Cells Have Distinct Transcriptional Signatures
A transcriptional analysis was undertaken to define a signature of gene expression that corresponded to WT and Foxk1–/–SP cell populations from unperturbed and injured skeletal muscle. Previous studies undertaken in our laboratory have rigorously characterized the isolation and amplification of RNA from limited ES cell numbers (100 to 1,000,000 cells), and we observed that these techniques were reproducible with minimal skewing of gene expression [19]. The respective SP cell populations were sorted, and RNA was isolated, amplified (two rounds), labeled, and hybridized to the Affymetrix oligonucleotide array (U74v2A-Chip; performed in duplicate). Using the MAS 5.0 expression report, the percent of probe sets present ranged from 37% to 43%, with the average signal intensity exceeding 725 and 3' to 5' ratios less than 4.5%. These results verify the integrity of the RNA samples and the assay quality for these studies. The analysis was performed with Affymetrix MAS5.0 Software to obtain detection calls of "present" (p < .04) or "absent" ( p > .06). A 95% confidence interval for fold change was constructed using standard errors of expression values. To define a molecular signature for SP cell populations, gene expression was compared to Affymetrix array analysis of RNA isolated and amplified from 3-day-old murine neonatal myoblasts [6] and ES cells (SM-1; passage 7). The respective cell numbers for each sample ranged between 3,000 and 5,000 cells. Gene expression for the respective cell populations was compared with a common denominator (i.e., irradiated STO fibroblast cells).

The analysis of the transcriptional profiling experiments revealed a discrete molecular program associated with the SMSP cell population. We confirmed the expression of Abcg2 in the SP cell populations using array technologies. Additionally, several transcripts expressed in endothelial cells (i.e., Vegf, Tie1, Vwf, Vcam1, Tie2/Tek, and Eng) were coexpressed in SMSP cells using Affymetrix array analysis. Several endothelial-restricted transcripts were absent in the SMSP cells, including the panendothelial marker CD31 (pecam1), the transmembrane ligand ephrinB2, and its receptor tyrosine kinase ephrinB4, which are molecular markers of embryonic arterial and venous endothelial cells, respectively (see online Tables 1Go and 2Go) [27–29]. Moreover, the expression of selected candidates was confirmed using RT-PCR analysis (Fig. 3BGo). Collectively, these results suggest either a common ancestry for these lineages (the precursor cell for SMSP and endothelial cells) or the use of shared signaling pathways between these cell populations.


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  		Table 1. Selected candidate gene expression (fold change) of BMSP, SMSP, and ES cells compared with a common denominator (i.e., STO cells)
 

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  		Table 2. Fold change of selected candidate transcript expression in the respective cell populations
 
As illustrated in the Venn diagram (Fig. 6Go and Table 1Go), the SMSP:BMSP:ES shared transcriptional program consists of 78 transcripts and broadly includes genes related to chemokines, cell cycle regulatory genes, metabolism, RNA processing, oxidative stress, protein degradation, and Notch signaling (Table 1Go and supplementary online Table 1Go). Other transcripts that are abundantly expressed in ES cells and absent in the SP cell populations include Esg1, Rex3, Oct4, Utf1, Sox2, and Nanog (Table 1Go and supplementary online Table 1Go) [19]. The SMSP:BMSP shared program consists of 467 transcripts (389 of these 467 transcripts are not shared with the ES cell population) related to nuclear proteins (Nmyc, Oct1, and Mef2A), channel proteins (e.g., chloride channel 3 and chloride channel 4), inflammation (colony stimulating factor 2, complement component 1, and cytokine inducible protein 2), cell-surface/adhesion proteins (fibrinogen-like protein 2, Icam, and Tyrobp), hematopoietic markers (Tie1 and Vwf), metabolism (Nd1, Pfkc, and lactotansferrin), stress (Hsp70 and thioredoxin reductase 1), and members of the Notch pathway (Table 1Go and supplementary online Table 1Go). Transcripts expressed only in the SMSP (483 transcripts) and not the bone marrow SP cells include nuclear factors (Meox2, Foxc1, Sox17, and Sox18), endothelial transcripts (adrenomedullin and Ang2), and cell-surface proteins (fibrinogen-like protein 2 and heparin sulfate) (Table 1Go). These results suggest that the molecular programs of the respective SP cell populations are more similar to each other than to the ES cell population. Furthermore, although the SP cell populations share several housekeeping transcripts, they have distinctive molecular programs.



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  		Figure 6. Transcriptional signatures of adult BMSP and SMSP are distinct from ES cells. Venn diagram of common and distinct molecular programs associated with BMSP, SMSP, and ES cells compared with a common denominator (i.e., STO cells) using Affymetrix array analysis. The number of transcripts significantly dysregulated are included in brackets. Abbreviations: BMSP, bone marrow side population; ES, embryonic stem; SMSP, skeletal muscle side population; WT, wild-type.

 
We additionally compared the molecular programs of Foxk1–/– SMSP, WT SMSP, and WT myoblasts (Fig. 7Go, Table 2Go). The Foxk1-null SMSP cell population shares several transcripts with the WT SMSP molecular program, and both of these programs (WT SMSP and Foxk1-null SMSP cells) differ considerably compared with the myoblast program (Fig. 7Go). The 269 transcripts that are distinctly expressed in the Foxk1 SMSP include MMPs (MMP8, MMP9, and complement factor H), schlaffen family members (schlafen 2 and schlafen 4), insulin growth factors (IGF2 and IGFBP5), and ectodermal neural cortex 1 (Table 2Go). Dysregulation of the schlaffen family members and insulin growth factors/binding proteins are consistent with the finding of perturbed cell-cycle regulation of the Foxk1 mutant SMSP cell population. Furthermore, the Foxk1-null SP cells have an induction of the hematopoietic molecular program (e.g., CD45, CD84, CD53, CD52, chemokine [C-C] receptor 2, chemokine [C-X-C motif] ligand 2, schlafen 4, and lipocalin 2), which may signify the recruitment of hematopoietic SP cells to skeletal muscle in the Foxk1-null mouse (online Table 3Go). In addition, we observed several transforming growth factor (TGF)-ß–responsive genes to be dysregulated in the Foxk1-null SP cells (Eng, Pdgfa, Enam, Thbs1, Cyp1a1, Vcl, Map3k1, and Tgfbi). Future studies will examine the role of Foxk1 in the regulation of the TGF-ß signaling pathway during embryogenesis and in the postnatal SP cell population. Both WT and Foxk1 mutant SMSP cells differ from myoblasts in their expression of Abcg2, endothelial markers (Ang2, endomucin, and endothelin 1), nuclear factors (Foxc1, Mef2A, and Meox2), muscle markers (MyoD, myogenin, myoglobin, troponin C, and skeletal {alpha}-actin), leukemia inhibitory factor receptor, and members of the Notch pathway (Table 2Go and supplementary online Table 1Go).



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  		Figure 7. Molecular signatures of Foxk1–/– SMSP, WT SMSP, and WT myoblasts. Venn diagram of common and distinct molecular programs of Foxk1-null SMSP, WT SMSP, and WT myoblasts compared with a common denominator (STO cells) using Affymetrix array technology. The number of dysregulated transcripts associated with the respective cell populations is included in brackets. Abbreviations: SMSP, skeletal muscle side population; WT, wild-type.

 

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  		Table 3. Fold changes of selected transcript expression in the respective cell populations
 
We observed that SMSP cells increase more than fourfold within 5 days after muscle injury. To additionally evaluate the increased SP cell population after muscle injury, we analyzed the transcriptional program of this cell population using array analysis (Fig. 8Go and Table 3Go). We observed that 314 transcripts were shared between the cell populations (i.e., SMSP isolated from unperturbed versus 5-day injured skeletal muscle), which were categorized using gene ontology annotation. The largest categories included transcriptional regulators, metabolism, and intracellular signaling (data not shown). Selected transcripts shared between these SP cell populations included nuclear factors (Sox7, Sox17, and Sox18), endothelial markers (protein C receptor, Vwf, Tie1, thrombomodulin, and Icam2) interleukins (interleukin-6), and members of the Notch and Wnt pathways (Table 3Go). The molecular program of the SMSP cells (5 days after injury) express limited numbers of muscle markers (skeletal {alpha}-actin, sarcoglycan epsilon, and myosin light chain alkali) and has decreased expression of proliferation markers (Pcna; Table 3Go). These results support the conclusion that SP cells participate in muscle repair.



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  		Figure 8. Molecular signature of WT SMSP isolated from regenerating skeletal muscle. Venn diagram of common and distinct molecular programs associated with BMSP, SMSP (isolated from normal adult skeletal muscle), and SMSP isolated from WT skeletal muscle 5 days after injury compared with a common denominator (i.e., STO cells). Note the number of significantly dysregulated transcripts that are indicated in brackets. Abbreviations: BMSP, bone marrow side population; SMSP, skeletal muscle side population; WT, wild-type.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Skeletal muscle has the capacity for repair and regeneration because of a rare population of MPCs or satellite cells that reside in adult muscle [1, 2]. These cells have an increased proliferative capacity and are capable of self-renewal [4]. A second population of cells has been isolated from skeletal muscle and several adult tissues that are enriched in repopulating cells and are referred to as SP cells [8]. Although several elegant studies have established the ability of the SP cells to contribute to alternative lineages, the molecular regulation of this cell population is ill-defined [14]. We have previously reported that the forkhead/winged helix transcription factor, Foxk1, is expressed in the MPC population in adult skeletal muscle and functions as a regulator of the cell cycle [5, 6].

In the present study, we pursued a comprehensive cellular and molecular analysis of the SP cell population isolated from normal, injured, and Foxk1 mutant skeletal muscle. These results enhance our understanding of the SP cell populations in response to chemical-induced muscle injury and their contribution to muscle repair in WT and Foxk1 mutant mice. Transcriptome analyses of these SP cell populations were additionally compared with ES cells, myoblasts, and bone marrow SP cells to define common and distinct molecular programs for the respective cell populations.

SP Cells Are Enriched in Repopulating Cells
SP cells were initially identified as a Hoechstlow subpopulation isolated from adult bone marrow using dual-wavelength flow cytometry [10, 11]. The ability of SP cells to efflux Hoechst 33342 dye is dependent on Abcg2, which is a member of the ABC transporters [12]. In the present study, we establish that the Abcg2 inhibitor, FTC, prevents the efflux of Hoechst dye by the SP cells. Furthermore, using RT-PCR techniques, Abcg2 is expressed in SP cell but not MP cell populations. SP cells have been isolated from many developing and adult tissues from several species [8, 9]. This cell population has been shown to be enriched in repopulating cells, because they have contributed to alternative lineages in studies using the murine model. In selected studies using genetically labeled cell populations, bone marrow SP cells have been reported to engraft and contribute to liver, skeletal muscle, and bone marrow regeneration [14, 30, 31]. In this study, we additionally establish that SMSP cells are capable of forming multinucleated myotubes in vitro and limited numbers of SMSP cells are able to compete with endogenous MPC or SP populations and contribute to the repair of injured, nonirradiated skeletal muscle. Collectively, these studies additionally support the conclusion that SP cells are enriched for stem cell activity. Future studies focused on clonal SP cell populations delivered into permissive environments will be necessary to define the multipotential capacity of this heterogeneous cell population [32].

SMSP cells have been implicated as a cell population that is distinct from the MPC (satellite cell) that resides in adult skeletal muscle [23]. Support for these two distinct populations has been obtained from the characterization of the Pax7 knockout mouse model. The Pax7 mutant skeletal muscle lacks MPC but contains an intact SP cell population and morphologically normal skeletal muscle [15]. Our studies additionally support the conclusion that SP and MPC are two distinct populations, because the SP cell marker, Abcg2, was absent in isolated fiber preparations but present in RNA isolated from vascular specimens. Using immunohistochemical techniques, we additionally established the localization ofAbcg2+ cells in the interstitial space and in close approximation to the skeletal muscle vasculature. Future studies will be required to further dissect the relationship between the SP and MPC populations in adult skeletal muscle.

Foxk1 Mutant Skeletal Muscle Has Decreased SP Cells
Selected members of the forkhead/winged helix transcription factor family have been shown to regulate stem cell and progenitor cell populations in the mouse, including Foxd3 (Genesis), Foxm1 (regenerating hepatocytes), and Foxb1 (also termed TWH, which is a regulator of neural progenitor cells) [33–35]. Additional members of this family have critical roles during embryogenesis, including the specification of cell fate, the regulation of patterning, and cellular proliferation [36–38]. Foxk1 is a member of the forkhead/winged helix transcription factor that has been previously shown to be expressed in the MPC population in adult skeletal muscle [5]. Using gene disruption strategies, we observed that Foxk1 knockout mice have severely impaired muscle regeneration attributable in part to decreased MPC numbers and perturbed cell-cycle progression (G1 arrest) of this population [6, 7]. In the present study, we observed that a subpopulation of the SMSP cells expresses Foxk1, and in the absence of Foxk1, the SMSP cells have dysregulated kinetics (i.e., numbers) after muscle injury. These results suggest that the Foxk1–/– SP cells are capable of responding to the signals and cues after injury but the decreased numbers may contribute to the impaired regenerative response.

Using emerging technologies, we examined the molecular program of the Foxk1-null SMSP cells. We observed an induction of chemokines, MMPs, and members of the Schlafen family (Slfn2, Slfn3, and Slfn4) in the Foxk1 mutant SMSP cells. Schlaffen family members have been reported to be potent inhibitors of cellular proliferation and arrest cells at the G0/G1 stage of the cell cycle [39]. Furthermore, insulin-like growth factor binding proteins are upregulated in the Foxk1–/– SMSP cell population and are associated with quiescent or inactive cell populations, which additionally support the arrested cell cycle in the absence of Foxk1 [40]. Moreover, the transcriptome analysis suggests that the TGF-ß pathway is perturbed in the absence of Foxk1. Future experiments will focus on Schlaffen family members, Igfbp, and TGF-ß as regulators of the SP cell cycle and candidate downstream target genes for Foxk1.

Molecular Program of the SP Cell Isolated from Normal and Injured Skeletal Muscle
In the present study, we compared the transcript expression of the cell populations to a common denominator (irradiated STO cells). Such an analysis allows for the enrichment of transcripts that are likely to reveal stem cell properties or stemness of the SMSP cell population [41]. Although the respective SP cell populations (skeletal muscle and bone marrow) were more similar to each other than they were to ES cells, all of the respective cell populations expressed transcripts involving the Notch pathway, cell-cycle regulatory genes, and Upp. The significance of Upp induction is unclear, although it seems to be consistently upregulated in cell populations with stem cell activity [41, 42].

SP cells have been identified and isolated from several embryonic and adult tissues. An outstanding question is whether the SP cells isolated from one lineage (i.e., skeletal muscle) are essentially the same as those isolated from other lineages (i.e., bone marrow). Previous studies suggest that SP cells isolated from different tissues differ in their cell-surface markers (i.e., Sca-1, c-Kit, and CD45). Our studies, using array technology, additionally support a distinct molecular program associated with the skeletal muscle and the bone marrow SP cells. We observed that the SMSP cells expressed several endothelial and hematopoietic transcripts, which support either a common ancestral precursor or shared pathways for the respective populations. To additionally examine the SP cell populations, we undertook an alternative analysis to generate a list of the transcripts associated with the respective cell populations based on absolute calls for present or absent expression (p < .05) [41]. Such an analysis reveals that the SMSP cells are Abcg2+, Sca-1+, c-Kit+, CD34+, and CD45-. The bone marrow SP cells differed from the SMSP in their expression of CD45+ (Abcg2+, Sca-1+, c-Kit+, CD34+, and CD45+). Furthermore, extensive analysis reveals the distinct molecular programs associated with each of these cell populations (supplementary online Table 2Go). We recognize that differences between protein and transcript expression exist and may be accentuated because of the use of enzymatic digestion (pronase) in the cellular dissociation, which may also cleave cell-surface proteins. The results of the present study can be compared with those of a recent study using a custom-designed cDNA microarray strategy to evaluate the transcriptome of an SP subpopulation of C2C12 myoblasts [43]. Although different chips were used in these two studies, the SMSP cell population and the SP C2C12 cell population commonly expressed Sca1, CD34, p57, cfos, Tgif, and Myc but lacked expression of CD45, Six, and Pitx2.

We observed a 4.5-fold increase in the SP cells isolated from injured skeletal muscle. This increase may be attributable to either an expansion of resident SMSP cells or, alternatively, a recruitment of SP cells from distant lineages (i.e., bone marrow). The increased expression of c-Kit and CD45 in the SP cell population isolated 5 days after cardiotoxin-induced injury suggests that a subpopulation of SP cells may be recruited from the bone marrow. Nevertheless, the increase in SP cells suggests that this cell population expands and repopulates injured skeletal muscle. This observation additionally supports the notion that SP cells have stem cell activity. Furthermore, we observed that SMSP cells isolated 5 days after injury had evidence of cell-cycle withdrawal and increased expression of muscle transcripts (but lacked induction of MyoD family members), consistent with the hypothesis that SP cells repopulate skeletal muscle. The lack of MyoD or myf5 expression in the SP cell population isolated after injury may be attributable to the limited time periods analyzed in this study. Further studies will be necessary to comprehensively analyze SP cell populations isolated at multiple, discrete periods after muscle injury.

Our studies further suggest that in response to a severe injury such as cardiotoxin, which destroys approximately 80% of the muscle, or in genetic mouse models that have impaired regeneration (Foxk1 mutant mice), SP cell populations may be recruited from extramuscular sources (i.e., bone marrow). Our studies suggest that SP cells (c-Kit+ and CD45+) isolated from injured skeletal muscle may be recruited from extramuscular sources, such as the bone marrow, or alternatively that this rare subpopulation that is resident in adult skeletal muscle expands significantly in response to a severe injury.

In summary, we provide data supporting the conclusion that SMSP cells increase in response to a severe injury and are capable of muscle differentiation. SP cell populations have distinct molecular signatures, and an enhanced understanding of these molecular regulatory pathways may stimulate novel biotechnological strategies to promote muscle regeneration in myopathic diseases such as muscular dystrophy.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We thank Sean Goetsch and John Shelton for their technical assistance and Dennis Bellotto for assistance with electron microscopy. We thank Dr. Susan E. Bates (National Institutes of Health) for the anti-Abcg2 serum and Dr. Lee Greenberger (Wyeth Research) for the FTC. We also acknowledge Drs. Cindy Martin, Eric N. Olson, Rhonda Bassel-Duby, and Beverly Rothermel for helpful discussions throughout the course of these studies. We thank Dr. Margaret A. Goodell (Baylor College of Medicine) and Shannon McKinney-Freeman for technical assistance and discussions throughout this study. This work was supported in part by grants from the Muscular Dystrophy Association (to A.P.M. and D.J.G.), the NIH (AR47850), and the Donald W. Reynolds Foundation.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Schultz E, McCormick KM. Skeletal muscle satellite cells. Rev Physiol Biochem Pharmacol 1994;123:213–257.[Medline]

  2. Hawke T, Garry DJ. Myogenic satellite cells: physiology to molecular biology. J Appl Physiol 2001;91:534–551.[Abstract/Free Full Text]

  3. Seale P, Rudnicki MA. A new look at the origin, function, and "stem-cell" status of muscle satellite cells. Dev Biol 2000;218:115–124.[CrossRef][Medline]

  4. Conboy IM, Rando TA. The regulation of notch signaling controls satellite cell activation and cell fate determination in postnatal myogenesis. Dev Cell 2002;3:397–409.[CrossRef][Medline]

  5. Garry DJ, Yang Q, Bassel-Duby R et al. Persistent expression of MNF identifies myogenic stem cells in postnatal muscle. Dev Biol 1997;188:280—294.[CrossRef][Medline]

  6. Hawke T, Jiang N, Garry DJ. Absence of p21 rescues myogenic progenitor cell proliferative and regenerative capacity in Foxk1 null mice. J Biol Chem 2003;278:4015–4020.[Abstract/Free Full Text]

  7. Garry DJ, Meeson AP, Elterman J et al. Myogenic stem cell function is impaired in mice lacking the forkhead/winged helix protein MNF. Proc Natl Acad Sci U S A 2000;97: 5416–5421.[Abstract/Free Full Text]

  8. Bunting KD. ABC transporters as phenotypic markers and functional regulators of stem cells. STEM CELLS 2002;20: 11–20.[Abstract/Free Full Text]

  9. Asakura A, Rudnicki MA. Side population cells from diverse adult tissues are capable of in vitro hematopoietic differentiation. Exp Hematol 2002;30:1339–1345.[CrossRef][Medline]

  10. Goodell MA, Brose K, Paradis G et al. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 1996;183:1797–1806.[Abstract/Free Full Text]

  11. Goodell MA, Rosenzweig M, Kim H et al. Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat Med 1997;3:1337–1345.[CrossRef][Medline]

  12. Zhou S, Schuetz J, Bunting K et al. The ABC transporter Bcrp1/Abcg2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 2001;7:1028–1034.[CrossRef][Medline]

  13. Zhou S, Morris J, Barnes Y et al. Bcrp1 gene expression is required for normal numbers of side population stem cells in mice, and confers relative protection to mitoxantrone in hematopoietic cells in vivo. Proc Natl Acad Sci U S A. 2002;99:12339–12344.[Abstract/Free Full Text]

  14. Gussoni E, Soneoka Y, Strickland CD et al. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 1999;401:390–394.[CrossRef][Medline]

  15. Seale P, Sabourin LA, Girgis-Gabardo A et al. Pax7 is required for the specification of myogenic satellite cells. Cell 2000;102:777–786.[CrossRef][Medline]

  16. Motoike T, Loughna S, Perens E et al. Universal GFP reporter for the study of vascular development. Genesis 2000;28:75–81.[CrossRef][Medline]

  17. Jackson KI, Mi T, Goodell M. Hematopoietic potential of stem cells isolated from murine skeletal muscle. Proc Natl Acad Sci U S A 1999;96:14482–14486.[Abstract/Free Full Text]

  18. Meeson AP, Radford N, Shelton JM et al. Adaptive mechanisms that preserve cardiac function in mice without myoglobin. Circ Res 2001;88:713–720.[Abstract/Free Full Text]

  19. Gallardo T, Hammer R, Garry DJ. RNA amplification and transcriptional profiling for analysis of stem cell populations. Genesis 2003;37:57–63.[CrossRef][Medline]

  20. Kim M, Turnquist H, Jackson J et al. The multidrug resistance transporter Abcg2 (breast cancer resistance protein 1) effluxes Hoechst 33342 and is over expressed in hematopoietic stem cells. Clin Cancer Res 2002;8:22–28.[Abstract/Free Full Text]

  21. Scharenberg CW, Harkey M, Torok-Storb B. The Abcg2 transporter is an efficient Hoechst 33342 efflux pump and is preferentially expressed by immature human hematopoietic progenitors. Blood 2002;99:507–512.[Abstract/Free Full Text]

  22. Litman T, Drule T, Stein W et al. From MDR to MXR: new understanding of multidrug resistance systems, their properties and clinical significance. Cell Mol Life Sci 2001; 58:931–959.[CrossRef][Medline]

  23. Asakura A, Seale P, Girgis-Gabardo A et al. Myogenic specification of side population cells in skeletal muscle. J Cell Biol 2002;159:123–134.[Abstract/Free Full Text]

  24. Gaca MD, Zhou X, Issa R et al. Basement membrane-like matrix inhibits proliferation and collagen synthesis by activated rat hepatic stellate cells: evidence for matrix-dependent deactivation of stellate cells. Matrix Biol 2003;22: 229–239.[CrossRef][Medline]

  25. Ward NL, Dumont DL. The angiopoietins and Tie2/Tek: adding to the complexity of cardiovascular development. Stem Cell Dev Biol 2002;13:19–27.

  26. Puri MC, Partanen J, Rossant J et al. Interaction of the Tek and Tie receptor tyrosine kinases during cardiovascular development. Development 1999;126:4569–4580.[Abstract]

  27. Shin D, Garcia-Cardena G, Hayashi S et al. Expression of ephrin B2 identifies a stable genetic difference between arterial and venous vascular smooth muscle as well as endothelial cells, and marks subsets of microvessels at sites of adult neovascularization. Dev Biol 2001;15:139–150.

  28. Gerety S, Anderson DJ. Cardiovascular ephrin B2 function is essential for embryonic angiogenesis. Development 2002;129:1397–1410.

  29. Kim I, Ryu YS, Kwak HJ et al. EphB ligand, ephrin B2, suppresses the VEGF and angiopoietin 1 induced Ras/mitogen-activated protein kinase pathway in venous endothelial cells. FASEB J 2002;16:1126–1128.[Abstract/Free Full Text]

  30. Majka SM, Jackson K, Kienstra K et al. Distinct progenitor populations in skeletal muscle are bone marrow derived and exhibit different fates during vascular regeneration. J Clin Invest 2003;111:71–79.[CrossRef][Medline]

  31. Wulf GG, Luo KL, Jackson KA et al. Cells of the hepatic side population contribute to liver regeneration and can be replenished with bone marrow stem cells. Haematologica 2003;88:368–378.[Abstract/Free Full Text]

  32. Jiang Y, Jahagirdar B, Reinhardt RL et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:41–49.[CrossRef][Medline]

  33. Sutton J, Costa R, Klyg M et al. Genesis, a winged helix transcriptional repressor with expression restricted to embryonic stem cells. J Biol Chem 1996;271:23136–23133.

  34. Ye H, Kelly T, Samandi U et al. Hepatocyte nuclear factor 3/forkhead homolog 11 is expressed in proliferating epithelial and mesenchymal cells of embryonic and adult tissue. Mol Cell Biol 1997;17:1626–1641.[Abstract]

  35. Dou C, Lee J, Liu B et al. BF-1 interferes with transforming growth factor beta signaling by associating with Smad partners. Mol Cell Biol 2000;20:6201–6211.[Abstract/Free Full Text]

  36. Lai E, Clark KL, Burley S et al. Hepatocyte nuclear factor 3/forkhead or "winged helix" proteins: a family of transcription factors of diverse biological functions. Proc Natl Acad Sci U S A 1993;90:10421–10423.[Abstract/Free Full Text]

  37. Xuan S, Baptista CA, Balas G et al. Winged helix transcription factor BF-1 is essential for the development of the cerebral hemispheres. Neuron 1995;14:1141–1152.[CrossRef][Medline]

  38. Winnier GE, Hargett L, Hogan BL. The winged helix transcription factor MFH1 is required for proliferation and patterning of paraxial mesoderm in the mouse embryo. Genes Dev 1997;11:926–940.[Abstract/Free Full Text]

  39. Schwarz DA, Katayama CD, Hedrick S. Schlafen, a new family of growth regulatory genes that affect thymocyte development. Immunity 1998;9:657–668.[CrossRef][Medline]

  40. Rousse S, Montarras D, Pinset C et al. Up-regulation of Insulin-like growth factor binding protein-5 is independent of muscle cell differentiation, sensitive to rapamycin, but insensitive to wortmannin and LY294002. Endocrinology 1998;139:1487–1493.[Abstract/Free Full Text]

  41. Ramalho-Santos M, Yoon S, Matsuzaki Y et al. "Stemness": transcriptional profiling of embryonic and adult stem cells. Science 2002;298;597–600.[Abstract/Free Full Text]

  42. Ivanova NB, Dimos JT, Schaniel C et al. A stem cell molecular signature. Science 2002;298:601–604.[Abstract/Free Full Text]

  43. Benchaouir R, Rameau P, Decraene C et al. Evidence for a resident subset of cells with SP phenotype in the C2C12 myogenic line: a tool to explore muscle stem cell biology. Exper Cell Res 2004:294:254–268.[CrossRef][Medline]

Received April 7, 2004; accepted for publication July 14, 2004.



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