Animals. All experiments were performed in accordance with the ethics code for animal experimentation. Ethical approval for all work was received from the animal research committee of Zhengzhou University (Zhengzhou, China). A total of 400 male C57BL/6J mice were purchased from Beijing HFK Bioscience Co., Ltd. (Beijing, China). The specific criteria for animal euthanasia included absence of food or water intake, low or no mobility, weak or absent heartbeat and absence of respiratory movement during the ongoing study, and all animals were euthanized at the end of the study. The mice were divided into different groups by sortition randomization method to control the cofounding bias. After intraperitoneal anesthesia (ketamine/midazolam 75 mg/kg and 5 mg/kg, respectively), mice were euthanized by cervical dislocation to minimize suffering. The method to confirm the death of mice included the absence of movement and heartbeat. All efforts were made to minimize suffering, including gaseous anesthesia with isoflurane (2-3%). Animals at 6-8 weeks of age (weight 20-25 g) were used for the experiments. Mice were kept in a pathogen-free environment at an invariable temperature and under a 12-h light-dark cycle. Pathogen-free chow and water sterilized using a high-pressure steam sterilizer were provided ad libitum.
MSC culture and expansion. MSCs isolated from the bone marrow of C57BL/6 mice were obtained from Cyagen Biosciences Inc. (Guangzhou, China). The identification of cells according to multipotency and the cell surface phenotypes was performed by the provider. The cell surface phenotypes were CD29+, CD44+, Sca-1+, CD117- and CD31-, characterized by fluorescence-activated cell sorting analysis（Fig.1）. The cells had the potential for differentiation into the osteogenic and adipogenic lineages as determined by staining with Alizarin red and oil red O, respectively. This evidence verified their identity as MSCs.
The mouse MSCs were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA), penicillin (100 U/ml), streptomycin (100 mg/ml) and 2 mM L-glutamine at 37°C in a humidified atmosphere containing 5% CO2. To obtain MSC clones, cells at 80-90% confluence were harvested and seeded into T25 flasks at 2x104 cells/cm2. Each clone was then picked and expanded. Cells at the 8th to 10th passages cultured in serum-free medium were used in the experiments.
Myocardial infarction and MSC transplantation. Mice were anesthetized by intraperitoneal injection of ketamine/medazolam (75 mg/kg and 5 mg/kg, respectively). With the application of a rodent ventilator, tracheal intubation was performed on the mice, a small thoracotomy was performed, and left anterior descending coronary artery (LAD) ligation was created as previously described (19), with a slight modification. A 10/0 Prolene suture was passed under the LAD at 1-1.5 mm distal to the left atrial appendage, immediately following the bifurcation of the major left coronary artery. At 1 h after surgical intervention, a total of 1x106 C57BL6/J MSCs in 25 µl complete medium were injected into five different points of the peri-infarct LV region in the MI group or the LV region in the sham group (20). Five points were separately injected with 2x105 MSCs in 5 µl medium. After 7, 14 and 28 days, TTC staining was performed to determine the infarct area of the LV. The remaining area of the LV was confirmed as the peri-infarct LV.
RT-qPCR for iNKT cells in post-MI hearts.
A total of 18 MI and 6 sham mice were created as described above. To observe time-dependent changes in iNKT cells, mice were sacrificed at 7, 14, and 28 days, and the LVs of their hearts were excised for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. As in a previous study (21), RT-qPCR for Vα14Jα18 (a specific marker of iNKT cells in C57BL/6 mice) was performed.
RT-qPCR for iNKT cells in post-MI hearts with MSC transplantation.
At 1 h after surgery, MI and sham mice were randomly assigned to one of the following groups: i) 25 µl serum-free medium (Me) alone, ii) 25 µl complete cell-free medium after cell culture (cMe), or iii) 25 µl complete medium with 1x106 MSCs injected into the myocardium of LV, with the suspension infused within 30 sec (22). Thus, there were six groups: Sham+Me (n=6), Sham+MSCs (n=6), Sham+cMe (n=6), MI+Me (n=18), MI+MSCs (n=18) and MI+cMe (n=18). After 7, 14 or 28 days, RT-qPCR analysis was performed to observe time-dependent changes in iNKT cells in the PLV area of the MI groups compared with those in the LV area of the sham groups.
To address the effective factors of cMe on the immunoregulation of MSCs, specific antagonists were used in the MI+cMe group: 20 µg/ml NG-monomethyl-L-arginine (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), an inhibitor of inducible nitric oxide (NO) synthase (iNOS), 5 µM indomethacin (indome; ApexBio, Glendale, CA, USA) for inhibition of prostaglandin E2 (PGE2), or 10 µg/ml anti-transforming growth factor (TGF)-β1 monoclonal antibody (R&D Systems Inc., Minneapolis, MN, USA), according to a previous study (8). In the fourth group, prostaglandin E2 (PGE2) was added to the MI+cMe groups. Each group included 6 mice. Subsequently, after 7 days, RT-qPCR analysis of Vα14Jα18 was performed (20).
RT-qPCR for iNKT cells, Masson staining and TUNEL staining in post-MI hearts with MSC treatment and iNKT cell activation.
Mice were randomly divided into five groups. At 1 h after MI, one group was injected with serum-free medium (Me) as a control, and another two groups randomly received an injection of 1x106 MSCs in 25 µl cMe with or without indome. In the fourth and fifth groups, α-galactosylceramide (α-GC; 0.1 µg/g body weight; Avanti Polar Lipids, Inc., Alabaster, AL, USA) was administered 30 minutes before MI specifically to activate iNKT cells while 1x106 MSCs with 25 µl complete medium were also injected into the sixth group. Therefore, there were six groups: Sham+Me (n=6), MI+Me (n=15), MI+MSCs (n=18), MI+MSCs+indome (n=18), MI+α-GC (n=18) and MI+MSCs+α-GC (n=18). After 7 and 28 days, RT-qPCR analysis of Vα14Jα18 was performed. After 28 days, the mice were euthanized, and the hearts were obtained. Tissue fibrosis of the LV was assessed by Masson’s trichrome staining, and apoptosis was evaluated by terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) and western blotting analysis for cleaved caspase-3 protein. Fibrotic and total areas of each image were measured by computerized planimetry (Image ProPlus 5.0, Media Cybernetics, Silver Spring, MD, USA), and the percentage of the fibrotic area was calculated as follows: (Fibrotic area/total area) x100%.
Methods of western blotting and TUNEL
Western blot analysis. Total protein was extracted from LV samples using IP lysis buffer after pulverizing in liquid nitrogen. The concentration of the protein was quantitatively measured by a BCA protein kit (Pierce, USA). The protein was mixed with loading buffer and boiled at 100°C for 10 min to denature it. Total protein (20 μg) was separated by 12% SDS-PAGE and transferred to 0.45 µm polyvinylidene fluoride (PVDF) membranes. After blocking with 5% nonfat-dried milk at room temperature for 1 h, membranes were incubated with primary antibodies [anti-cleaved caspase-3 antibody (Cat. No. ab49822; Abcam; USA) and anti-GAPDH antibody (Cat. ab181602 Abcam; USA)] overnight at 4°C and then incubated with horseradish peroxide-conjugated secondary antibodies at room temperature for 1 h. The blots were developed with ECL reagent (Millipore, Germany). GAPDH was used for normalization. The sum density of each protein band was measured by ImageJ software.
TUNEL. The mouse heart LV samples were dehydrated by standard graded alcohol solutions and embedded in paraffin as described previously (PMID: 31419728). LV samples in paraffin were sectioned longitudinally into 5 μm thin sections. After three washes with PBS (pH = 7.4), the sections were incubated with proteinase K for 30 min at 37°C. After treatment with cell permeable fluid (Servicebio, G1204), the sections were assayed using an in situ death detection kit (Roche, 11684817910) according to the manufacturer's instructions. All of the sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Finally, the numbers of total cells and TUNEL-positive cells were counted in at least three noncontinuous fields of each specimen using an inverted fluorescence microscope (Zeiss, Germany). The percentage of positive cells (positive cells/total cells ×100) was determined as the apoptotic rate (%). Cell counting was performed by a pathologist blinded to the experimental conditions.
Statistical analysis. Data analysis was performed using SPSS software 18.0 for Windows (SPSS, Inc., Chicago, IL, USA). Values are expressed as the mean ± standard deviation. Each experiment was performed at least three times independently. Analysis of variance and ANOVA were performed for multiple and two-group comparisons. P<0.05 was considered to indicate a significant difference.