Oxidative stress preconditioning of mouse perivascular myogenic progenitors selects a subpopulation of cells with a distinct survival advantage in vitro and in vivo

Cell engraftment, survival and integration during transplantation procedures represent the crux of cell-based therapies. Thus, there have been many studies focused on improving cell viability upon implantation. We used severe oxidative stress to select for a mouse mesoangioblast subpopulation in vitro and found that this subpopulation retained self-renewal and myogenic differentiation capacities while notably enhancing cell survival, proliferation and migration relative to unselected cells. Additionally, this subpopulation of cells presented different resistance and recovery properties upon oxidative stress treatment, demonstrating select advantages over parental mesoangioblasts in our experimental analysis. Specifically, the cells were resistant to oxidative environments, demonstrating survival, continuous self-renewal and improved migration capability. The primary outcome of the selected cells was determined in in vivo experiments in which immunocompromised dystrophic mice were injected intramuscularly in the tibialis anterior with selected or non-selected mesoangioblasts. Resistant mesoangioblasts exhibited markedly enhanced survival and integration into the host skeletal muscle, accounting for a more than 70% increase in engraftment compared with that of the unselected mesoangioblast cell population and leading to remarkable muscle recovery. Thus, the positive effects of sorting on mesoangioblast cell behaviour in vitro and in vivo suggest that a selection step involving oxidative stress preconditioning may provide a novel methodology to select for resistant cells for use in regenerative tissue applications to prevent high mortality rates upon transplantation.


Introduction
The release of several types of factors, such as cytokines and growth factors, from damaged tissues stimulates both resident and circulating stem cells to initiate tissue repair programmes. [1][2][3] However, the therapeutic efficacy of stem cells is compromised by reduced homing towards the target site 4,5 and by the cytotoxic environment, which causes massive cell death during the first several days post-transplantation. [5][6][7][8][9] For this reason, enhancing in vivo stem cell viability may be a key step in improving the outcomes of cell-based therapies. The microenvironment within damaged tissues is unfavourable for stem cell survival due to hypoxia, inflammatory mediators, a lack of glucose or serum and oxidative stress, with the latter being particularly detrimental. 6,10,11 In particular, hydrogen peroxide (H 2 O 2 ), a reactive oxygen species (ROS) that diffuses freely into and out of cells, 12,13 may play a significant role in inducing the apoptosis or necrosis of injected stem cells. [13][14][15] Although the regulation of cell death by external oxidative stress has been extensively studied in vitro, these experiments typically use differentiated cells rather than stem cells and focus on events that occur shortly after treatment (i.e., a few minutes later or at most in the first 24 h). 16,17 In the field of stem cell research, in vitro experiments based on comparative analyses of oxidative stress resistance among mesenchymal stem cells, embryonic stem cells and induced pluripotent stem cells (iPSCs) have shown that iPSCs and embryonic stem cells are less resistant to oxidative stress than mesenchymal stem cells. 18 However, other studies have demonstrated that oxidative stress induces senescence in human mesenchymal stem cells. [19][20][21] Therefore, despite its central role in the development of cell-based therapies, the effects of exogenous oxidative stress on stem cell viability are not well understood. To explore the reasons why only a few stem cells survive after transplantation, we first performed an in vitro study. H 2 O 2 was used to apply extreme exogenous oxidative stress to mouse mesoangioblast perivascular myogenic progenitors (hereafter referred to as 'mabs' or 'A6 cells') to isolate resistant cells that survived after a long recovery period. The resistant cells (hereafter 'cell clones' or 'H2 cells') exhibited the unusual ability to retain self-renewal capacity in addition to increased migratory and proliferation capabilities with respect to the untreated mab population. Moreover, in vivo experiments involving the intramuscular injection of cell clones into immunocompromised dystrophic mice further highlighted noteworthy improvements in cell survival, migration and engraftment into host skeletal muscle tissue compared with those of unstressed cells. Mabs are easily expandable in vitro and have largely been studied for cell-based therapeutic applications; thus, they are prime candidates for skeletal muscle regeneration and reconstruction. [22][23][24][25][26][27] Therefore, mabs derived from the selected subpopulation are better able to tolerate oxidative stress and display distinct survival and integration advantages in vivo upon transplantation, representing an important approach to potentiate improvements in mab-based cell therapy.

Different H 2 O 2 doses and exposure times affect mab cell cycle progression and viability
To select resistant cells that survive in an oxidative environment, we identified a sub-lethal concentration of H 2 O 2 that inhibited cell cycle progression and partially killed the treated cells. We analysed the dose responses of   . 1a) and 50% cell survival (Fig. 1b), representing optimal conditions to isolate oxidative stress-resistant mabs. Cell cycle analysis by cytofluorimeter revealed higher G 2 /M phase arrest after exposure for 24 h of but not at shorter time points (i.e., 4, 8, or 14 h), indicating that arrest was strictly dependent on the length of exposure (Fig. 1c). This high dose was the maximum theoretical H 2 O 2 concentration achievable in tissues with severe inflammation. Thus, the highest tolerable concentration that blocks mitosis and does not immediately induce complete death in mabs was chosen to select for the oxidative stress-resistant subpopulation.
We also investigated the p38 activation pathway, which may participate in cell cycle arrest during mitosis (G 2 /M phase) due to Cdc25B inhibition resulting from increased phosphorylated p38 MAPK activity. 28,29 To explore whether this same mechanism occurs in mabs upon oxidative stress exposure (400 μM H 2 O 2 ), we evaluated p38 MAPK levels after H 2 O 2 exposure for different periods of time (4,8,14 and 24 h) when we observed mitotic blockade (see Fig. 1c). Western blotting (Figs. 1d, e) showed that both alpha-and beta-phosphorylated isoform levels increased during oxidative stress at these same time points when we observed mitosis blockade. This association appeared functionally relevant because mabs treated with both 400 μM H 2 O 2 and a p38 MAPK inhibitor (SB202190) did not exhibit G 2 /M phase blockade (Fig. 1f). This finding supports a role for p38 MAPK activity in cell cycle blockade due to oxidative stress.

Cell viability, proliferation and ROS generation during the recovery period
To study long-term survival in H 2 O 2 -treated mabs, specific analyses were performed during the recovery period. Throughout recovery, a portion of the treated cells died, as assessed by trypan blue exclusion assays. This trend began to reverse on the eighth day of recovery (Fig. 2a). Simultaneously, during the 1 days of recovery, the cells were still blocked in G 2 /M phase (Fig. 2b); cell cycle distribution returned to normal on day five. Thus, although the cell cycle was restored, cell death continued daily. The intracellular ROS concentration was maximal on the 1 day of recovery, indicating that at the end of treatment, the stressed cells were producing large quantities of endogenous ROS (Fig. 2c).  . All data were obtained from three independent experiments. Error bars indicate standard deviation. *P < 0.5, **P < 0.05, ***P < 0.005 vs control group, or H 2 O 2 group clones (H2, E3, B9), lower percentages of cells were blocked in G 2 /M phase after treatment than that of mabs (Supplementary Figs. S1A-D). The three cell clones were similar, and for the remainder of the study, we carefully analysed the H2 clone to determine whether it preserved the mab phenotype while possessing enhanced resistance to oxidative stress.

The H2 clone retains the basal mab phenotype
To determine whether the H2 cell clone retained the mab phenotype under normal growth conditions, we assessed morphology, cell doubling time, intracellular ROS levels, expression patterns of certain stemness genes, and differentiation capabilities. In general, there were no differences between mabs and the H2 cell clone among the examined parameters (Supplementary Figs. S2A-C and Fig. 3a); the exception was the myogenic differentiation of the H2 cell clone, which demonstrated an improved ability to form myotubes in co-culture with the C2C12 mouse myoblast cell line (Fig. 3b). To evaluate the myogenic capabilities of selected and unselected mabs, lentiviral infection was performed to label the cells with nuclear β −galactosidase (nLacZ) 26,30 , and the effective participation of the mab nuclei in myotube formation was evaluated, revealing a remarkable increase in H2 myogenic activity compared with that of its A6 counterpart (Fig. 3c).

Oxidative stress does not affect H2 clone viability, proliferation and migration capacity
The sorted cell clone exhibited a behavioural alteration in the presence of an oxidative environment. The cell clone and mabs were subjected to severe oxidative stress using 200 μM H 2 O 2 for 24 h; this concentration may be similar to that encountered in vivo in chronically inflamed tissues. According to a cell cycle analysis, this lower H 2 O 2 dose arrested mabs in G 2 /M phase (Fig. 4a) during oxidative stress and recovery, as did the higher dose (400 μM) (see Fig. 2b). In contrast, the cell clone was not arrested in G 2 / M phase, resulting in further analyses (Fig. 4b). We next evaluated the effects of H 2 O 2 on the viability of both A6 and H2 cells by analysing the cell percentages in different stages of apoptosis using flow cytometry after performing Annexin V-PE/SYTOX Green double staining, which distinguishes between different stages of apoptotic cell death. After H 2 O 2 treatment, both cell lines showed a marked increase in apoptosis, although the apoptosis levels  were higher in A6 than H2 cells (Fig. 4c), confirming the superior resistance of H2 cells to oxidative stress. Then, we characterized the growth capacity of this cell clone, which was doubled compared to the total cell number on day one of recovery after stress induction (Fig. 4d). Conversely, the total number of mabs was reduced, suggesting that mabs and the cell clone behaved differently after exposure to oxidative stress. The proliferative responses of both mabs and H2 cells were also observed after CFSE labelling ( Fig. 4e) and quantitatively evaluated using a CFSE-based cytometry assay. After different time points, cell proliferation was assessed by processing the degree of reduction in green fluorescence intensity using ModFit LT TM software (Fig. 4f). As shown in Fig. 4g, H2 cells had a higher proliferation index than A6 cells under basal growth conditions. A significant reduction in this index was observed 24 h after treatment with 200 μM H 2 O 2 . Despite this reduction, H2 cells exhibited a proliferation index higher than that of A6 cells one day after H 2 O 2 removal. Both H2 and A6 cells demonstrated proliferative capacities comparable to pre-stressed conditions. The H2 proliferative index was doubled after 2 days of recovery compared with that of A6 cells.
Another fundamental stem cell function in transplantation applications is migration capacity; therefore, we evaluated the H2 cell clone for its expression of matrix metalloproteinase 2 (MMP2), which is crucial for migration 31 , to define H2 migratory capacity compared with that of unselected mabs. Real-time PCR and western blot analyses revealed increased MMP2 expression in the H2 cell clone (Fig. 5a-c). To determine whether this increased MMP2 expression was related to enhanced migration  . e Fibres Cross Sectional Analysis (CSA) highlights the muscle recovery due to H2 effect on αSGKO dystrophic muscle, presenting the disappearance of small (under 500 μm 2 ) degenerating muscle fibres capability, we performed a scratch test that revealed effective, increased and rapid cell clone migration under normal growth conditions compared with that of mabs (Fig. 5d, e). We then simulated an in vivo scenario in an in vitro assay and repeated the scratch test in the presence of 200 µM H 2 O 2 (Fig. 5d, e). The H2 cell clone maintained more rapid migration than treated mabs during the first 24 h of continuous oxidative stress. Based on these results, the H2 cell clone subpopulation was more resistant to an oxidative environment.

In vivo transplantation of H 2 O 2 -preconditioned mabs
To verify the improved survival and migration capacity of the H2 cell clone, in vivo experiments were performed via the intramuscular injection of cells into an immunocompromised, dystrophic mouse model: SCID/alpha sarcoglycan null mice. 32 H2 clone cells and unselected wildtype mabs were modified to express nLacZ, and 5 × 10 5 cells were implanted into the tibialis anterior (TA) muscles of the same mice. The muscles were then collected for real-time PCR (Fig. 6a) and immunohistochemical analysis (Figs. 6b, c) 48 h and 20 days, respectively, after injection. RNA was isolated from the treated TAs, and real-time PCR revealed a remarkable increase in LacZ expression related to the numbers of surviving and engrafted mabs upon implantation (Fig. 6a). βgalactosidase immunostaining also revealed notably high levels of LacZ-positive nuclei in the muscles injected with the H2 cell clone (Fig. 6b d, e) compared with that in the contralateral TA muscles treated with unselected mabs (Fig. 6b a, b, Supplementary Fig. S3). Moreover, high magnification revealed greater spreading within the injected TAs and remarkable integration of H2 cell clone nuclei into regenerating myofibres labelled by laminin immunostaining, reflecting peripheral LacZ nuclei inside host muscle fibres (arrows in Supplementary Figs. S3 and arrows in Figs. 6b, f). We speculate that the higher numbers of lacZ-positive cells observed when H2 cell clones were injected into the TA primarily reflects cell survival; unsorted mabs injected into the dystrophic environment were less resistant and had a lower survival rate (Fig. 6c). Moreover, mice treated with intramuscular H2 cells exhibited a notable ameliorated muscle morphology characterized by more uniform myofibre size and the absence of inflammatory infiltration and degenerating-regenerating areas marked by small fibres, which are clear signs of dystrophy pathology (Fig. 6d). Further supporting this morphofunctional muscle recovery, cross sectional area (CSA) analysis of myofibres clearly revealed the amelioration of muscle fibres upon H2 cell injection, presenting uniform dispersion of fibre size within the recovered muscle; this was also reflected in the recovery of alpha sarcoglycan expression in mice treated with H2 cells (Fig. 6e).

Discussion
The current study reports that a mouse mesoangioblast stem cell line, A6, is a heterogeneous population. This finding, together with the ability to select clones exhibiting particular behaviours after severe oxidative stress, may have important implications. The results obtained in vitro and in vivo suggest that the use of selected cells demonstrating improvements in specific activities is preferable to using the entire population when addressing issues related to tissue repair. Intra-heterogeneity is an important aspect of stem cell biology, as reflected in human mesenchymal stem cells, which are highly complex at the biochemical level. 33 In some cases, certain clones derived from a stem cell population have more abundant transcripts than the parental population, and vice versa. Clonal and microarray analyses have shown that cardiac progenitors expressing Mesp1 are distinct populations, most likely due to their regional segregation during early gastrulation. 34 Here, we isolated a subpopulation from mesoangioblast vessel-associated progenitor cells derived from the mouse embryonic dorsal aorta at stage E9.5. 22,25,35 The selected subpopulation demonstrated excellent survival following severe oxidative stress for 24 h, while cell death occurred for the majority of treated mabs. Published in vitro results for human mesenchymal stem cells have revealed premature cell senescence upon H 2 O 2 treatment 19-21 and permanent growth blockage. 36 Pomduck and colleagues 37 improved mesenchymal stem cell resistance to oxidative stress by generating cells with specific protein overexpression profiles. Here, we proposed a selection step to isolate oxidative stress-resistant cells from a heterogeneous population and demonstrated its major advantages. Our results provide reliable evidence supporting the idea that selected cells-or the H2 cell clone-resist cell cycle arrest and are more proliferative. Moreover, the H2 clone migrates faster than mabs in scratch assays both under physiological growth conditions and oxidative stress, based on the observed increased MMP2 expression in these cells, and possess a greater capacity to differentiate. More importantly, H2 cells transplanted into a mouse model of muscular dystrophy were able to considerably ameliorate muscle morphology and restore alpha sarcoglycan expression.
One lingering question is whether surviving cells became resistant due to the negative effects of oxidative stress or due to alterations that occur prior to stress. A study of mesenchymal stem cells demonstrated that specific H 2 O 2 concentrations induce cell differentiation via the upregulation of Notch signalling, which promotes cell differentiation upon oxidative induction. 38 In our case, oxidative stress triggered this change/resistance in only a few cells, specifically a subpopulation of mabs, indicating the heterogeneous nature of the starting mab population. Thus, it is possible to promote the cell survival, migration and differentiation capacity of a subpopulation of cells by preconditioning stem cells with an oxidative stress signal, subsequently leaving unaltered or even ameliorating the original capabilities of the preconditioned cells.
In conclusion, our findings highlight the potential of stem cell clone isolation as a therapeutic modality for use in regenerative medicine.

Cell culture and treatment
Mabs were grown on type I collagen treated plates in DMEM (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum and 1% antibiotic and antimycotic (Life Technologies) in a humidified 5% CO 2 atmosphere at 37°C. To induce oxidative stress, cells were grown in medium with H 2 O 2 (Sigma, St. Louis, MO, USA) at different concentrations. Mabs were treated with H 2 O 2 only after reaching the confluence of 80%. We used SB 202190 (10 µM for 30 min) (Sigma) as p38 inhibitor.

Trypan blue dye exclusion
To measure viability, cells were stained with trypan blue (Sigma) dye and then counted as described elsewhere. 39

Cytofluorimetric analysis
For cell cycle analysis cells were collected and washed with phosphate buffered saline (PBS) (Life Technologies). Pellets were treated with 0.1% C 6 H 5 Na 3 O 7 , 0.1% NP-40, 50 μg/ml propidium iodide (Life Technologies) and 0.06 mg/ml RNase A (Sigma) for 45 min at RT in the dark. The cells were acquired on a BD FACSCanto (BD Biosciences, Franklin Lakes, NJ, USA) using FACSDiva Software. The analysis was performed using Flowing software 1.6.0.
To determine the intracellular ROS content, cells were collected, washed with PBS and incubated with 2′,7′dichlorfluorescein-diacetate (Sigma) 5 µM for 30 min at 37°C. The pellet that had been previously washed with PBS was resuspended again in PBS. Cells were acquires on a BD FACSCanto and analyzed using Flowing software 1.6.0.

Preparation of protein extracts
Protein extracts were prepared as previously described. 31 Briefly PBS-washed cells were resuspended in lysis buffer (20 mM Hepes, pH 7.9; 0.4 M NaCl; 0.2 mM EDTA; 10% glycerol; 0.5 mM dithiothreitol; 0.5 mM PMSF; 1X protease/phosphatase inhibitors, Sigma) and lysed by three cycles of freezing (liquid nitrogen) and thawing (37°C water bath). Cell debris was removed by centrifugation at 17,000 rpm for 30 min at 4°C.
The extract protein concentrations were determined by the Bradford microassay method (Sigma) using bovine serum albumin as a standard.

Adipocyte differentiation assay
Cells were treated with 10 ng/ml of dexamethasone (Sigma) in complete medium for 5 days and then analyzed for adipocyte morphology. Cells were washed with PBS, and after were fixed with formaldehyde 10% (Sigma) for 90 min. Cells were washed three times with PBS and were then incubated with oil red O (Sigma) for 30 min. After cells are washed with PBS and observed.

Scratch (wound) assays
Scratch assays was performed as previously described. 31 Confluent monolayer of mabs and cell clone was wounded by using a 200 µl pipette tip. Cells were washed with DMEM (Life Technologies) (without serum and antibiotics), to remove any detached cells and photographs of the wound were taken. Treatment with H 2 O 2 200 µM (Sigma) was added after wound creation. After 3, 6, 12 and 24 h the cells were photographed. All images were made at RT in the medium in which the cells were grown, using a contrast phase microscope (Leica DMIL LED, Wetzlar, DE) with a 4X (HIPLAN NA. 0.10, Leica) objective, magnification 40X and a camera Cool Snap ProColor (Media Cybernetics, Rockville, MD, USA). Four representative extents of cell migration near the wound areas were photographed.

Quantitaive RT-PCR (qRT-PCR)
Total RNA was extracts with an RNeasy Mini Kit (Qiagen, Hiden, DE), according to the manufacturer's instructions. qRT-PCR was performed on MMP2. GAPDH and actin were used as internal controls. All reactions were performed by using the i-Script One Step RT-PCR Sybr Green Kit (BioRad) according to the manufacturer's instructions. The real time RT-PCR primers are summarized in Table 1. qRT-PCR for MMP2 was performed in a total of 25 µl from a mixture containing 0.5 µl of Taq Polymerase with Sybr green mix, 12.5 µl of 2X Buffer, 4.5 µl of forward primer (900 nM), 4.5 µl of reverse primer (900 nM), 1000 ng of RNA for MMP2 gene. Reactions were run in BioRad CFX-96 detector system under the following conditions: 40 cycles of 95°C for 10 s, 60°C for 30 s, after pre-incubation at 95°C for 5 s. All reactions were performed in triplicate. The specificity of the amplification reactions was confirmed by meltingcurve analysis. The fold changes in the mRNA expression level of MMP2 in the two cell lines and control sample were compared using the DDCq method (BioRad CFX Manager Software).
Real time RT-PCR for in vivo mabs injection was performed as described. 40 Briefly, total RNA was extracted by liquid nitrogen homogenization with TRIzol reagent (Life Technologies) following the manual instruction. The obtained RNA was cleaned and DNase treated with RNeasy Mini Kit (Quiagen). Single-stranded cDNA was synthesized with SuperScript First-Strand RT-PCR system (Life Technologies), according to the protocol supplied by the manufacturer. Real time PCR was performed using SYBR GREEN PCR Master Mix (PE Applied Biosystems Foster City, CA, USA) according to the supplied method by intron-spanning primers:

Mesoangioblast trasnsduction with lentiviral vectors
Third-generation lentiviral vectors encoding nuclear β-Galactosidase were employed for mabs transduction, mabs expressing nuclear LacZ (Mabs-nLacZ) were cultured and analyzed in vitro by myogenic fusion and differentiation, and in vivo by intramuscular injection. 26,30 Animals and treatments Three-months-old SCID (Severe Combined Immune Deficiency) Alpha Sarcoglycan (α-SG) null mice were used for intramuscular injection. 31 Briefly mice were anesthetized with an intramuscular injection of physiologic saline buffer (10 ml/kg) containing ketamine (5 mg/ ml) (Bayer, Leverkusen, DE) and xylazine (1 mg/ml) (Bayer). For intramuscular cell delivery, approximately 5 × 10 5 Mabs-nLacZ were injected into the TA via a 0.20 mm diameter needle inserted along the cranio-caudal axis TA. Mice were sacrificed at different time points for molecular and morphological analysis.

Immunohistochemistry
The tissue samples were embedded in O.C.T. (BioOptica, Milano, IT) and flash-frozen in liquid-N 2 for 15 sec. Sections were cut at a thickness of 8 μM using a Leika cryostat. The obtained sections were hydrated with PBS and stained with X-Gal (Thermofisher, Waltham, MA, USA) to reveal β-galactosidase positive cells as described; 26,30 briefly sections were washed with PBS (two wash, 5 min each) and incubated 24 h at 37°C with 'X-Gal working solution'. X-Gal working solution is composed of 'X-gal stock solution' (X-Gal 40 mg/ml in N,N-dimethyl formamide stored at −20°C and light protected) diluted 40X in the 'X-Gal dilution buffer' (Potassium Ferricyanide Crystalline 5 mM, Potassium Ferricyanide Trihydrate 5 mM, Magnesium Chloride 2 mM in PBS, light protected and stored at 4°C). The sections were then incubated with primary antibody (Rabbit anti-Laminin from SIGMA at a 1:100 final concentration) diluted with blocking buffer (PBS with 0.2% Triton X-100 and 20% heat-inactivated goat serum) for 20 min at room temperature (RT). Sections were washed with washing solution (PBS with 0.2% Triton X-100 and 1% Bovine Serum Albumine) and then incubated with secondary antibody (HRP-conjugated goat anti-rabbit from Chemicon, diluted 1:500 in 20% goat serum). The immune reaction was developed using 3-amino-9 ethylcarbazole substrate (AEC, Sigma). Or for H&E staining, sections were washed with PBS (two wash, 5-10 min each), counterstained with aqueous Eosin (Sigma) and then covered directly with aqueous mounting medium, Aqua Poly/ Mount (Polysciences, Inc., Warrington, PA, USA) The LacZ positive nuclei were counted for three different nonadjacent transverse sections from the largest TA portion for 3 mice per experimental group.

Immunofluorescence
Immunofluorescence was performed according with previous publication 41 . Briefly, the cell cultures were fixed in 2% paraformaldehyde (Sigma) for 15 min at 4°C, then washed with wash buffer (PBS with 0.2% Triton X-100) and incubated with primary antibodies diluted with blocking buffer (PBS with 0.2% Triton X-100 and 20% heat-inactivated goat serum) for 1 hr at RT. The primary antibodies used for cell culture staining were mouse anti Myosin Heavy Chain (MF20) (Developmental Studies Hybridoma Bank, Iowa city, IA) at 1:2 dilution and rabbit anti-lacZ (Cappel) diluted 1:100. For histological section, collected TA were treated as described previously for sectioning then sections were rehydrated in PBS and soaked in blocking buffer (PBS with 0.2% Triton X-100 and 20% heat-inactivated goat serum) for 20 min at RT. Afterwards the sections washed with washing buffer and incubated with primary antibodies diluted with blocking buffer. the primary antibody used were: rabbit anti-Laminin from SIGMA at a 1:100 final concentration and mouse anti-αSarcglycan (Vector, Ad1/20A6) diluted at 1:100. After several washes with washing buffer, sections were incubated with secondary antibodies diluted with blocking buffer for 1 hr at RT. The secondary antibodies used at 1:500 were goat anti-mouse FITC (Chemicon, Waltham, MA, USA) and goat anti-rabbit Alexa488 (Molecular Probes). Specimens were counterstained with DAPI (1:500, Molecular Probes) 10 minutes at RT to detect nuclei, washed several times with wash buffer and photographed with a Nikon Eclipse TE2000 microscope equipped with a CoolSNAP-MYO CCD camera (Photometrix, Kew, AUS) and MetaMorph software (Molecular Devices, Sunnyvale, CA, USA).

Cross-sectional area analysis
Cryosections stained with anti-laminin antibody were analyzed using an ImageJ macro which automatically detects single fibres within the laminin fluorescent signal. Wrongly detected fibres were manually corrected before to start the CSA measurement. For each sample, the area of more than two thousand single fibres was measured.

Statistical analysis
Unpaired two-tailed student's t-test for comparison of two independent groups were performed with GraphPad software (GraphPad Software Inc., La Jolla, CA, USA). Quantitative data are represented as the mean ± standard deviation or SEM of at least three independent experiments.