CXCL12 and osteopontin from bone marrow-derived mesenchymal stromal cells improve muscle regeneration

Muscle satellite cells are essential for muscle regeneration. However, efficient regeneration does not occur without muscle-resident mesenchymal progenitor cells. We show here that bone marrow-derived mesenchymal stromal cells (Bm-MSCs) also facilitate muscle regeneration in Duchenne muscular dystrophy (DMD) model mice. Bm-MSCs transplanted into peritoneal cavities of DMD model mice with severe muscle degeneration strongly suppressed dystrophic pathology and improved death-related symptoms, which resulted in dramatic lifespan extension. Isolated single myofibers from Bm-MSC-transplanted mice manifested considerably less myofiber splitting compared with myofibers from non-transplanted mice, which indicated that transplantation significantly ameliorated abnormal regeneration. With regard to the number of satellite cells, several cells remained on myofibers from Bm-MSC-transplanted model mice, but satellite cells rarely occurred on myofibers from non-transplanted mice. Also, CXCL12 was crucial for muscle regeneration. CXCL12 facilitated muscle regeneration and paired box protein–7 (PAX7) expression after cardiotoxin-related muscle injury in vivo. The majority of primary muscle satellite cells sorted by integrin-α7 and CD34 expressed CXCR4, a receptor specific for CXCL12. CXCL12 strongly suppressed p-STAT3 expression in these sorted cells in vitro. CXCL12 may therefore influence muscle regeneration through STAT3 signaling in satellite cells. Targeting these proteins in or on muscle satellite cells may improve many degenerative muscle diseases.

flask. CIM was replaced every 3-4 days. After 2 weeks, cells were lifted by incubation with 0.25% trypsin/1 mM EDTA for 2 minutes at 37°C. These cells (passage 2) were then expanded by plating at 50 cells/cm 2 in complete expansion medium (CEM) consisting of Iscove's modified Dulbecco medium (IMDM; Invitrogen) supplemented with 9% FCS, 9% HS, 100 U/ml penicillin (Invitrogen), 100 µg/ml streptomycin (Invitrogen), and 12 µM L-glutamine (Invitrogen). CEM was replaced every 3-4 days. After 10 days, these cells (passage 3) were lifted by incubation with 0.25% trypsin/1 mM EDTA for 2 minutes at 37°C. Passage 3 cells were either frozen or expanded further by plating at 50 cells/cm 2 and incubating them in CEM. For freezing, cells were resuspended at 3.0 × 10 5 cells/ml in 5% DMSO and 95% FBS, frozen at −80°C for 24 hours, and then stored in liquid nitrogen. To recover frozen cells, a vial was quickly thawed to 37°C, and cells were plated in a 175-cm 2 flask in CEM and incubated for 2 days. After 2 days, the flask was washed with PBS, and cells were lifted by incubation with 0.25% trypsin/1 mM EDTA for 2 minutes at 37°C, plated at 50 cells/cm 2 in CEM, and incubated for 12 days, with the medium changed every 3-4 days. The same conditions were used for successive passages.
For adipogenesis, cultures were incubated in IMDM that was supplemented with 10% FCS, 10% HS, 100 U/ml penicillin, 100 µg/ml streptomycin, 12 mM L-glutamine, 5 µg/ml insulin (Sigma), 50 µM indomethacin (Sigma), 1 × 10 -6 M dexamethasone, and 0.5 µM 3-isobutyl-1-methylxanthine (Sigma). The medium was changed two times per week for 3 weeks. Cells were fixed in 10% formalin for 20 minutes at room temperature and were then stained with 0.5% Oil Red O (Sigma) in methanol (Wako, Osaka, Japan) for 20 minutes at room temperature. anti-CD11b (clone M1/70, catalog MAB1124; R&D Systems); anti-CD45 (clone 30-F11, catalog FAB114F-025; R&D Systems); isotype control rat IgG2A (clone MAB006; R&D Systems); isotype control rat IgG2B (clone MAB0061; R&D Systems); and secondary developing reagents (R&D Systems). We followed the manufacturer's instructions. In brief, Bm-MSCs cultured in CEM were lifted with trypsin/EDTA and resuspended in Flow Cytometry Staining Buffer at a concentration of 1.0 × 10 6 cells/ml. Cells were added with each antibody or with isotype-matched control antibody (10 µl in 100 µl), followed by incubation for 30 minutes at 4°C. After incubation, cells were washed twice and resuspended in the same buffer. Resuspended cells were added to a secondary developing reagent according to the manufacturer's instruction, followed by incubation for 30 minutes at 4°C in the dark. Cells were washed twice and resuspended in the same buffer for flow cytometry. 45 . We explanted single myofibers for culture from the left and right calf muscles (soleus and gastrocnemius) from C57BL/10 (wild-type) mice. To analyze myofiber appearance and the number of satellite cells (SCs) on myofibers, single fibers were isolated from dko mice and dko/MSC mice. For myofiber isolation and culture, culture medium must contain 110 mg/ml sodium pyruvate so that fibers survive. Mice were killed by using cervical dislocation, and muscles were removed immediately by microdissection, with care being taken to handle muscles only by their tendons to minimize damage to the fibers. The removed muscles were rinsed with PBS, were put into a 25-ml centrifuge tube with a triple seal cap (Iwaki, Tokyo, Japan) containing 5 ml of fresh filter-sterilized 0.2% (w/v) type I collagenase (Worthington Biochemical, Lakewood, NJ) in DMEM plus GlutaMAX-I (Gibco, Grand Island, NY) supplemented with 2 mM L-glutamine (Gibco), and were incubated in a CO2 incubator at 37°C for 1.5-2 hours.

Isolation of single myofibers and their culture
After the muscles were digested, samples were transferred, with 5-3/4-inch Pasteur pipettes (Iwaki), to the first of a series of 50 mm × 20.3 mm plastic Petri dishes (Thermo Fisher Scientific) containing 20 ml of DMEM. To prevent fibers from sticking to the Petri dishes and the pipettes, the former were prerinsed and the latter were preflushed with 5% BSA (Sigma) in PBS. We used a transilluminating stereo dissecting microscope to liberate single muscle fibers by repeated flushes of medium with the 5-3/4-inch Pasteur pipettes. After 20-30 intact, viable single muscle fibers were separated, the muscle bulk was transferred to a fresh Petri dish, and the previous dish, containing separated fibers and "debris" (hypercontracted fibers, fiber pieces, fiber clusters, and collagen wisps), was incubated in a humid environment at 37°C and 5% CO2. We repeated the fiber separation cycle until we had dissociated sufficient numbers of fibers from the muscles. Intact, viable muscle fibers were then separated from debris by transferring them with a normal Pasteur pipette to a fresh Petri dish, in which they were incubated until plating. Before plating, wells of 6-well tissue culture plates were briefly rinsed with 50% Matrigel (Becton, Dickinson, Franklin Lakes, NJ), which was applied ice-cold to prevent premature gelation, and plates were then incubated for 30 minutes at 37°C. Individual muscle fibers were removed from suspension with a 9-inch Pasteur pipette (Iwaki), and each fiber was placed in the center of a well with 2 ml of plating medium. Plating medium consisted of DMEM plus GlutaMAX-I supplemented with 2 mM L-glutamine and 10% HS. During the first 3 days, the fibers were cultured in plating medium. On the fourth day, culture medium was changed to proliferation medium, which consisted of DMEM plus GlutaMAX-I supplemented with 2 mM L-glutamine, 10% HS, 20% FBS, and 1% (v/v) chick embryo extract (United States Biological, Salem, MA). The medium was changed every 3 days. One day before coculture, Bm-MSCs (1.0 × 10 5 cells) were added to each 30-mm cell culture plate insert, Preset VECELL (Vessel, Kitakyushu, Japan), and were maintained in CEM. When single fibers were placed in 6-well culture plates, they were immediately covered by culture inserts containing Bm-MSCs. After the reactions, products were separated on 1.0% agarose gel.
CXCL12 expression vector construction and CXCL12 preparation. As we mentioned above, the forward primer contained a Kozak consensus sequence for efficient translation. The amplified product was first cloned into the pCR-Blunt II-TOPO (Thermo Fisher Scientific). After we confirmed the sequence, CXCL12 cDNA carrying the Kozak sequence was re-cloned into pcDNA (pRc/RSV; Invitrogen) mammalian expression vector and was named pcDNA-CXCL12. The expression vector was purified from the large-scale culture by means of the Plasmid Giga Kit (QIAGEN, Tokyo, Japan).

pcDNA-CXCL12 was transfected into HEK293 cells cultured in 10% FCS-containing
DMEM by the conventional calcium phosphate precipitation method. After precipitation, cells were cultured overnight, and then they were washed thoroughly with PBS to remove precipitates and expression vector, after which they were cultured as usual in 10% FCS/DMEM for 1 more day to wait for efficient CXCL12 transcription. On the third day after transfection, to remove FCS from the culture, cells were washed repeatedly with PBS followed by changing the culture medium to a serum-free medium. After 2 more days of culture, we collected the medium that we expected contained CXCL12 secreted from transfected HEK293 cells.
We quantified the mouse CXCL12 concentration by using ELISA for mouse-specific CXCL12. We also quantified human CXCL12 from HEK293 cells, to see whether it was present, by using human-specific ELISA, but human CXCL12 was below the detectable concentration.
As we mentioned above, we prepared mouse-specific CXCL12 under serum-free conditions. When we used CXCL12 in vivo and in vitro, supernatant of serum-free medium collected from the mock-transfected HEK293 cells was used as a negative control.

CXCL12 concentration in culture medium of Bm-MSCs as determined by ELISA.
Bm-MSCs were expanded by plating at 50 cells/cm 2 in CEM consisting of IMDM (Invitrogen) supplemented with 9% FCS, 9% HS, 100 U/ml penicillin (Invitrogen), 100 µg/ml streptomycin (Invitrogen), and 12 µM L-glutamine (Invitrogen). After 2 days of culture, we removed FCS from the culture and washed the cells repeatedly with PBS.

Muscle SC isolation and culture
Kinex Antibody Microarray. We isolated SCs by using FACS, harvested 1.0 × 10 5 cells, and plated them in 6-well tissue culture plates with 50% Matrigel (Becton, Dickinson).
We cultured the cells in medium that consisted of DMEM plus GlutaMAX-I supplemented with 2 mM L-glutamine, 10% HS, and 20% FBS and incubated them in a 5% CO2 incubator at 37°C for 6 days. We changed this medium and added CXCL12 every 5 days. On day 6, we performed the protein array studies by using used FACS to isolate muscle SCs, which were CD34 + and integrin-α7 + . About 70% of these cells were also CXCR4 + . (b) We incubated muscle SCs for 4 days, followed by immunofluorescence staining, which allowed identification of SCs, as this representative image shows (nucleus, red, arrowheads; PAX7, green, arrows). Scale bar: 100 µm. (c) We incubated muscle SCs for 7 days and observed them with the All-in-One Fluorescence Microscope BZ-9000 (Keyence). Scale bar: 100 µm.