Animals and cell culture
Fresh skeletal muscle was obtained from 8-week-old Sprague-Dawley rats. Rat tibialis muscles were dissociated by collagenase type I (0.2% collagenase, Sigma), then were filtered through a 40 μm filter (Biosharp). After isolation, myofiber-associated cells were subjected to flow cytometry analysis (see below). Markers for CD45 (BD Pharmingen), CD11b (BD Pharmingen), anti-integrinβ1 (ab95622, Abcam) and CD34 (ab81289, Abcam) were used. After sorting, cells were cultured in DMEM supplemented with 20% FBS and 1% chick embryo extract. Myogenic differentiation was induced using DMEM supplemented with 2% heat-inactivated horse serum.
Fresh skeletal muscle (20 g) was obtained from 8-week-old adult Sprague-Dawley rats. Samples were decellularized at room temperature according to the following procedures: shaking in 1% SDS for 48 hours (method A), shaking in 0.2% sodium deoxycholate for 48 hours followed by shaking in 1% SDS for16 hours (method B), shaking in 1% Triton X-100 for 36 hours followed by shaking in 1% SDS for 36 hours (method C), shaking in 0.1% ethylenediaminetetraacetic acid (EDTA) for 6 hours followed by shaking in 0.2% sodium deoxycholate for 8 hours (method D), shaking in 0.2% trypsin/0.1% EDTA for 36 hours followed by shaking in 0.5% Triton X-100 at for 48 hours (method E), or shaking in 0.1% EDTA for 6 hours followed by shaking in 1% SDS for 12 hours (method F). Then, we removed the remaining nuclear materials by shaking in 9×106 U/l DNase-I (Thermo Fisher Scientific, USA) and 6×107 U/l RNase (Yami Biotechnology Co., Ltd., Beijing) for 24 hours. Finally, residual reagents were removed from the samples by repeated washes with deionized water and centrifugation.
Samples were fixed in 4% paraformaldehyde for 24 hours and then processed according to the manufacturer’s instructions. Staining with hematoxylin and eosin (H&E), Masson’s trichrome and 4’,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA) was performed. Images of the samples were obtained using an Axio Observer Z1 inverted microscope (Carl Zeiss, Inc., Germany) with an Axiovision 4.8 camera.
Preparation of decellularized rat skeletal muscle matrix hydrogel and assessment of its properties
To prepare the decellularized skeletal muscle matrix hydrogel, 10 mg of ECM powder was put into 900 µl pepsin (Sigma-Aldrich) solution (0.01 M HCl, pepsin: ECM = 1:10) and then incubated under shaking for 48 hours until dissolved. After NaOH was added to stop the reaction. Then, phosphate-buffered saline (PBS) was added and evenly mixed and the mixture was incubated at 37°C for 30 min, 2 hours, and 24 hours for in vitro gelation. The matrix solution (500 µl) was placed at 37°C for gelation and assessed every 20 min. The ECM hydrogel was disinfected under ultraviolet light and its thickness was very thin.
Quantitative analysis of the DNA content, protein content and growth factors
The DNA content of fresh skeletal muscle, ECM and hydrogel was analyzed by a dsDNA Assay Kit (Invitrogen Inc.). To evaluate the protein content in the three kinds of samples, a bicinchoninic acid (BCA) protein detection kit (Sigma-Aldrich) was adopted. A Blyscan GAG Assay Kit (Biocolor) and rat enzyme-linked immunosorbent assays (ELISAs) (Minneapolis, MN) were used to quantitatively identify the levels of sulfated glycosaminoglycans (s-GAG) and fibroblast growth factor (bFGF).
Scanning electron microscopy
All samples were prepared according to the manufacturer’s instructions prior to analysis via scanning electron microscopy. Sample cross-sections were analyzed by a scanning electron microscope (Quanta 250, FEI) at an accelerating voltage of 30 kV.
Flow cytometry analysis and cell sorting were performed at the central laboratory of The First Affiliated Hospital of Sun Yat-sen University. Both sorting and analysis were carefully performed according to the manufacturer’s instructions.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA extraction and amplification were performed according to the manufacturer’s instructions. The following primers were used: Pax7: forward 5′-AGCCGAGTGCTCAGAATCAA-3′ and reverse 5′-TCCTCTCGAAAGCCTTCTCC-3′, MyoD: forward 5′CGACTGCCTGTCCAGCATAG-3′ and reverse 5′-GGACACTGAGGGGTGGAGTC-3′, MyHC: forward 5′-TGCCAAGACCGTGAGGAATG-3′ and reverse 5′-AATGCATCACAGCTCCCGTG-3′, and GAPDH: forward 5′-GGGTGATGCTGGTGCTGAGTATGT-3′ and reverse 5′-AAGAATGGGAGTTGCTGTTGAAGTC-3′.
Cell viability analysis
MTT assay was performed according to the manufacturer’s instructions. SkMSCs were seeded in 96-well plates (1 × 104 cells/ml) coated with or without decellularized skeletal muscle ECM hydrogels. The concentration of solubilized MTT formazan was measured by a microplate reader (TECAN, Austria) at an absorbance wavelength of 490 nm.
Cell proliferation assay
A 5-ethynyl-2′-deoxyuridine (EdU) kit (RiboBio, Guangzhou) was adopted to evaluate the proliferation of SkMSCs. Briefly, SkMSCs were seeded in 6-well plates (8×105 cells/well) coated with decellularized skeletal muscle ECM hydrogels and cultured in growth medium and then sorted into the following four treatment groups: NC group, TGFβ1 group (2.5 ng/ml TGFβ1), TGFβ1 + PD98059 group (2.5 ng/ml TGFβ1 + 10 µM PD9809) and PMA group (25 nmol/L phorbol 12-myristate 13-acetate (PMA).
Immunofluorescence staining and microscopy
Cultures were fixed with 4% paraformaldehyde, processed for single and double
immunofluorescence and stained with DAPI to identify the nuclei. Primary antibodies against PAX7 (1:100 dilution, ab199010, Abcam, USA), MyoD (1:250 dilution, ab203383, Abcam), myosin heavy chain (1:200 dilution, ab11083, Abcam, USA), anti-laminin (ab11575), anti-collagen IV (ab6586), and anti-desmin [Y66] cytoskeletal markers (ab32362) (1:100 dilution, ab129002, Abcam, Cambridge, MA, USA) were used. Matching secondary antibodies conjugated to goat anti-mouse IgG H&L (1:1000 dilution, Alexa Fluor® 594) (ab150116) and goat anti-rabbit IgG H&L (Alexa Fluor® 488) (1:1000 dilution, ab150077, Abcam) were used. Cell nuclei were counterstained with DAPI (ab228549, Abcam, USA). Cell images were obtained by an Axiovision 4.8 camera and an Axio Observer Z1 inverted microscope (Carl Zeiss, Inc., Germany).
Confocal electron microscopy
Cells were seeded on confocal dishes and maintained in an incubator for 24 hour. The procedures were performed according to the manufacturer’s instructions. The primary antibodies included Pax7 (1:200 dilution) and MyoD (1:200 dilution), which were then incubated with goat anti-mouse IgG H&L (1:1000 dilution, Alexa Fluor® 594) (ab150116) and goat anti-rabbit IgG H&L (Alexa Fluor® 488) (1:1000 dilution, ab150077, Abcam) for 1 hour in the dark at room temperature. Then, DAPI was added and a confocal microscope (Nikon) was used to acquire images.
Dual-luciferase reporter assay
The luciferase reporter assay was performed using the Dual-Luciferase Reporter Assay System (Promega). Cells were transfected with pmiGLO-based luciferase vector fused or not fused to the wild-type or mutated integrin–3′UTR. METTL3 or empty vectors were cotransfected.
RNA stability assay
Actinomycin D (Sigma-Aldrich) was added to SkMSCs at 5 mg/ml to assess RNA stability. After incubation for indicated time points, the cells were collected and RNA samples were extracted for reverse transcription and qPCR. mRNA transcription was inhibited with Actinomycin D and the degradation rate of RNA (K decay) was estimated.
Statistically significant differences were determined by t-tests for comparisons between two groups and by one-way analysis of variance (ANOVA) for comparisons between more than two groups. All data were analyzed using SPSS 22.0 software.