Animals and ethical statement
All animal experiments were approved by the Institutional Ethics Committee of the Second Affiliated Hospital of Zhejiang University School of Medicine. Male Sprague–Dawley (SD) rats (two months old, 250 ± 10 g) were purchased from Slac Laboratory Animal Co., Ltd. (Shanghai, China) and were housed in a temperature-controlled (22 ± 1 °C) and humidity-controlled (60 ± 5%) room under a 12-h light–dark cycle. The animals had free access to food and water.
ESC-MSCs preparation and identification
ESC-MSCs were differentiated from ESC using a two-step process. In brief, H9-ESC colonies (YuanSheng Biotech Corporation, Hangzhou, China) were dissociated into small clumps after 3 min of incubation with TrypLE Express and then transferred to ultralow-attachment plates in E8 media (Gibco, Grand Island, NY, USA). After 7 days, embryoid bodies (EBs) were harvested and plated in MSC induction medium composed of Dulbecco’s modified Eagle’s medium (high glucose), 10% fetal bovine serum, and 1 mM l-glutamine. After 2 w, the EB outgrowths were subcultured using TrypLE Express. These cells were ESC-MSCs and were designated passage 0 (P0). The differentiated ESC-MSCs attained a homogenous population of spindle-shaped cells. Passage 4 (P4) ESC-MSCs were used in the following animal experiment.
In this study, phenotypic identification of MSCs was performed using flow cytometry (BD Biosciences, USA) with the following monoclonal antibodies: anti-CD73-PE-Cy7, anti-CD90-APC, anti-CD105-PE, anti-CD14-APC, anti-CD34-PE, anti-CD45-fluorescein isothiocyanate (FITC), CD79a-PE, and HLA-DR-PE. All antibodies were purchased from BD Pharmingen (China). After antibody labeling, data were acquired using an Agilent NovoCyte and analyzed using NovoExpress.
Establishment of the ACS animal model
The ACS model was established by infusing isotonic normal saline as previously described [8, 9]. In brief, the rats were anesthetized by intraperitoneal administration of 3% pentobarbital sodium. Once the animals were anesthetized, a 24-gauge angiocatheter was inserted into the anterior compartment of the left hindlimb in the experimental group to elevate compartment pressure. The intracompartmental pressure was increased to 80 mmHg and maintained between 80 ± 10 mmHg for 2 h, and a single-incision fasciotomy was performed to decompress the compartment at the end of the experiment. Sham animals underwent all procedures, but the compartment pressure was maintained at the baseline level (0 mmHg). Rats were sacrificed by an intra-arterial overdose (1.5 ml) of 3% pentobarbital sodium, and blood, lung, and tibialis anterior (TA) muscle samples were harvested for further processing.
Experimental design
This study consisted of three separate parts (Fig. 1). A total of 168 rats were used in this study.
Part 1
To investigate the therapeutic effects of ESC-MSCs on ACS-induced skeletal muscle injury, rats were randomly divided into four groups: the Sham + PBS, Sham + MSCs, ACS + PBS, and ACS + MSCs groups at different time points (1 day, 3 days, and 7 days). After fasciotomy, ESC-MSCs (1.2 × 106 in 0.5 ml of PBS) or an equal volume of PBS was administered to the rats by the dorsal penis vein. The muscle edema index and serum creatine kinase (CK) level were measured at 1 day, 3 days, and 7 days after ACS. Moreover, enzyme-linked immunosorbent assay (ELISA) and hematoxylin and eosin (H&E) staining were performed 1 day after ACS. Terminal dUTP nick end labeling (TUNEL) staining and western blotting were performed 3 days after ACS. Additionally, rat ethology was tested 7 days after model induction, and H&E and Masson trichrome staining of the TA muscle was performed 7 days after ACS.
Part 2
To clarify whether macrophages polarization was involved in ESC-MSC therapy, rats were randomly assigned to two groups: the ACS + PBS and ACS + MSCs groups at different time points (0 h, 1 day, 3 days, 5 days, and 7 days). Cells were labeled with Paul Karl Horan fluorescent dye (PKH26) before being intravenously injected to track the distribution of ESC-MSCs in vivo. Western blotting and immunofluorescence analysis was performed at 0 days, 1 day, 3 days, 5 days, and 7 days after ACS. Moreover, quantitative real-time PCR was performed 3 days after ACS.
Part 3
To identify the role of macrophages in ESC-MSC therapy, macrophages were depleted in rats by intravenous injection of liposomal clodronate (LC). Sham rats were injected with the same amount of liposomal vehicle (LV). Serum CK analysis, H&E staining, quantitative real-time PCR, immunohistochemistry, and western blotting were performed 3 days after macrophage depletion and ESC-MSC therapy.
ESC-MSCs labeling
For ESC-MSC labeling, we incubated the cells with PKH26 (10 μM) (Sigma, St Louis, MO, USA) at 37 °C for 5 min. After the cells were washed with PBS three times, ESC-MSCs were diluted to a concentration of 2.4 × 106 cells/ml in PBS for injection.
Macrophages depletion
For the macrophages depletion studies, rats were intravenously injected with 1 ml of (5 mg/ml) LC (Vrije Universiteit, Amsterdam, Netherlands) 1 day prior to and 1 day following ACS injury as previously described [26]. Rats injected with LV (Vrije Universiteit) were used as the control.
Blood sampling and serum analysis
Blood samples were collected from the abdominal aorta, and serum was obtained by centrifugation. Serum CK levels were measured using a commercial CK kit (Jiancheng Bioengineering Institute, Nanjing, China). The concentrations of tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6) and interleukin 10 (IL-10) were measured using ELISA kits (Elabscience, Wuhan, China). The optical density (OD) was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, MA, USA).
Muscle edema index
The left and right TA muscles were harvested and weighed immediately, and the left/right TA muscle weight ratio was calculated to determine the muscle edema index.
Rat ethology
The hanging grid test and grip strength test were used to assess skeletal muscle function. The hanging grid test was performed as described previously for mice [27]. The rats were placed individually at the center of a wire mesh screen (2 mm wire thickness). The screen was suspended 50 cm above a plastic cage filled with sawdust bedding, and the grid was inverted with the head declining first. The hanging duration was recorded in three independent trials conducted at least 20 min apart. The data from all three trials were averaged.
The grip strength test was performed using a grip strength meter (Handpi HP-5N, Shenzhen, China) as previously described [28]. The rats were held by the tail and approached the grid slowly until the rat's hind claws grasp the grid, and were gently pulled by the tail until they released their grip. The forces of three trials were recorded and averaged.
Histological analysis
The TA muscles were fixed in 10% formalin for 24 h and underwent routine dehydration and paraffin embedding. Tissue sections (4 µm) were stained with H&E or Masson trichrome and examined under a light microscope (Leica, Germany).
H&E staining was performed to evaluate the pathological damage to skeletal muscle at 1 day and skeletal muscle regeneration at 7 days after ACS. The histological damage score was determined using 5 random fields selected from each sample at an objective magnification of 20× as previously described [29]. Briefly, sections from each tissue were scored for tissue damage by two investigators in a blinded fashion, but only the highest score was considered. Disorganization and degeneration of the muscle fibers (0: normal, 1: mild, 2: moderate, 3: severe); and inflammatory cell infiltration (0: normal, 1: mild, 2: moderate, 3: severe). Regenerative myofibers were identified as those containing central nuclei [30]. Under an objective magnification of 20×, five random fields were selected from each sample and used to quantify the total number of regenerative myofibers. To measure the diameters of regenerative myofibers, the minor axis diameters of the regenerative myofibers were measured in each TA muscle as previously described [31].
Masson trichrome staining was performed to measure fibrosis 7 days after ACS. The fibrotic area of skeletal muscle was quantitated using 5 random fields selected from each sample at an objective magnification of 20×. ImageJ software was used to calculate the percentage of the fibrotic area.
Skeletal muscle fiber injury in Part 3 was evaluated 3 days after macrophages depletion and ESC-MSC therapy. The injury score was determined based on a protocol established by McCormack et al. [32]. Four random fields of each H&E-stained section at an objective magnification of 20× were examined, and based on the proportion of injured cells (defined by ragged cellular edges, vacuolation, lymphocyte infiltration, or rhabdomyolysis), a numerical value between 0 and 10 was determined.
TUNEL and dystrophin staining analysis
Apoptotic nuclei in skeletal muscle were examined using double-fluorescent labeling of TUNEL and dystrophin. TUNEL staining was performed according to the manufacturer’s protocol (Roche Inc., Basel, Switzerland). After TUNEL labeling, tissue sections were incubated with a rabbit anti-dystrophin monoclonal antibody (1:200, Cat. 12715-1-AP, Proteintech) followed by an anti-rabbit IgG cyanin 3 (Cy3) (1:200, Cat. SA00009-2, Proteintech). Sections were incubated with DAPI (Meilunbio, Dalian, China) to stain nuclei and examined under a fluorescence microscope (Olympus, Tokyo, Japan). Photomicrographs were merged and saved by Image-Pro Plus software (Olympus). The numbers of TUNEL- and DAPI-positive nuclei were counted and only labeled nuclei that colocalized with dystrophin staining were counted. The data are expressed as the TUNEL index, which was calculated by counting the number of TUNEL-positive nuclei divided by the total number of nuclei. The TUNEL index for each muscle was calculated from five random, nonoverlapping fields at an objective magnification of 40×.
Immunofluorescence staining
Immunofluorescence staining was conducted as previously described [33] to detect M1 macrophage (CD86) and M2 macrophage (CD206) infiltration in skeletal muscle. Skeletal muscle sections were subjected to antigen retrieval, and the sections were blocked and labeled overnight at 4 °C with rabbit anti-CD86 (1:200, Cat. 13395-1-AP, Proteintech) and mouse anti-CD206 (1:200, Cat. sc-58986, Santa Cruz). After the sections were washed in PBS three times, FITC- or Cy3-labeled secondary antibodies (1:200, Proteintech) were used for the final immunostaining. Sections that were not incubated with primary antibodies were used as negative controls. The sections were incubated with DAPI (Meilunbio) to stain nuclei and examined under a fluorescence microscope (Olympus).
Western blotting
Western blotting was used to determine protein expression as previously described [34]. TA muscle tissue was lysed in RIPA lysis buffer (Biosharp, Hefei, China). After centrifugation, soluble proteins were quantified with a BCA kit (Biosharp) and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The proteins were electrophoresed until sufficiently separated and then transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were blocked with 5% nonfat dry milk in Tris-buffered saline, and then the PVDF membranes were incubated with the following primary antibodies: rabbit anti-tubulin (1:5000, Cat. #5335S, CST), rabbit anti-cleaved caspase3 (1:1000, Cat. #9664S, CST), rabbit anti-caspase3 (1:1000, Cat. #9662S, CST), rabbit anti-Bax (1:1000, Cat. #14796S, CST), rabbit anti-Bcl-2 (1:1000, Cat. #3498S, CST), rabbit anti-CD86 (1:1000, Proteintech), mouse anti-CD206 (1:1000, Santa Cruz), and rabbit anti-CD68 (1:1000, sc-20060, Santa Cruz) at 4 °C overnight. Then, the PVDF membranes were incubated with corresponding horseradish peroxidase (HRP)-conjugated IgG antibodies at room temperature for 2 h. Bands were visualized using an ECL kit (Millipore, Billerica, MA, USA). The band densities were quantified with ImageJ software (NIH).
Quantitative real-time PCR
RNA was extracted and analyzed using a previously described method [31]. Total RNA was obtained with TRIzol reagent (Invitrogen, MA. USA) and quantified by a Nanodrop spectrophotometer (Thermo Fisher). RNA was then reverse transcribed by the PrimeScript RT reagent kit (Yeason, Shanghai, China). Then, quantitative real-time PCR was performed with SYBR Mixture (Yeason), specific rat primers and cDNA using the Mx3000P real-time PCR system (Agilent Technologies, USA). β-Actin was used as the internal reference. The sequences of primers were used as follows: rat β-Actin forward: 5′-TGTCACCAACTGGGACGATA-3′, reverse: 5′-GGGGTGTTGAAGGTCTCAAA-3′; rat TNF-α forward: 5′-ATGGGCTCCCTCTCATCAGTTCC-3′, reverse: 5′-GCTCCTCCGCTTGGTGGTTTG-3′; rat IL-6 forward: 5′-ACTTCCAGCCAGTTGCCTTCTTG-3′, reverse: 5′-TGGTCTGTTGTGGGTGGTATCCTC-3′; rat IL-10 forward: 5′-CTGCTCTTACTGGCTGGAGTGAAG-3′, reverse: 5′-TGGGTCTGGCTGACTGGGAAG-3′;
Immunohistochemistry
Immunohistochemistry was performed as previously described [35]. Skeletal muscle sections were subjected to antigen retrieval, and the sections were blocked and labeled overnight at 4 °C with rabbit anti-CD68 (1:200, Santa Cruz). After being incubated with HRP-conjugated secondary antibodies (Boster, Wuhan. China), the sections were treated with an avidin–biotin–peroxidase conjugate (Boster). The reaction was visualized using diaminobenzidine (DAB) substrate chromogen solution (Vectorlabs, CA, USA) after the tissue was counterstained with hematoxylin.
Statistical analysis
The data are expressed as the means ± standard deviation (SD). Statistical and graph analyses were performed using GraphPad Prism 8.0 (GraphPad Software, USA). For two-group comparisons, significance was assessed by Student’s t-tests. For multiple group comparisons, significance was assessed by One-way ANOVA with Tukey’s post hoc test, two-factor ANOVAs with Bonferroni pair-wise comparisons. Values of P < 0.05 were considered statistically significant.
