All experiments involving mice were approved by the Institutional Animal Care and Use Committee of Tongji University (Shanghai, China; approval no: TJLAC-019-131). The human umbilical cords were obtained from full-term births and implemented following the approval of Ethic Committee and the informed consent of donors in Shanghai East Hospital.
HucMSCs were isolated and cultured in good manufacturing practices (GMP) lab. In this experiment, the culture procedures of primary HucMSCs were as follows. The umbilical cord was placed in the 10 cm dish and cut into 2-3 cm tissue. Blood from the umbilical cord was washed and umbilical vein and artery were removed. The Wharton’s jelly was separated from the umbilical cord and cut into pieces (approximately 1 cm). Then, pieces of tissue were tiled and cultured in Minimum Essential Medium, Alpha (α-MEM, Corning, 10-022) supplemented with 5% UltraGRO-Advanced (Helios, HPCFDCGL50), and were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2 until HucMSCs slowly crawled out of pieces. After that, the medium was changed every 2-3 day, and HucMSCs underwent passages using Trypin-Express (Gibco, 12604021) solution. HucMSCs were used at passage 4-5 (P4-5) for all experiments.
Tumorigenicity evaluation of HucMSCs in vivo
To confirm the tumorigenicity of HucMSCs in vivo, nude mice were randomly (using computer-generated random numbers) divided into two groups named HucMSC and A549 groups, and injected subcutaneously into the left arm with 1*106 HucMSCs or A549 cells, respectively. The survival situation of each group was evaluated for 120 days.
Multiple enzymes detection
After HucMSCs intramyocardial injection, blood (about 300 μL) from different groups was collected on day 7 and 28, and the serum was isolated by centrifugation. Aspartate transaminase (AST), Alanine aminotransferase (ALT), Creatine (Cr), Lactate dehydrogenase (LDH), Creatine kinase (CK), Creatine kinase isoenzyme (CK-MB) levels were measured using Beckman AU680 (Beckman Coulter, Inc.) according to manufacturer’s instructions.
After euthanizing animals, mice tissues were fixed in 4% paraformaldehyde (PFA) overnight, embedded in paraffin and sectioned at 3-4 μm intervals. The sections were stained with Hematoxylin and eosin (H&E) and Masson’s Trichrome. For Masson’s Trichrome, fibrosis was quantified as the relative area of blue staining compared the left ventricle surface, as an average of six sections per heart at the level of the papillary muscle, using Image J software (National Institutes of Health).
MI model establishment and HucMSCs injection
The 8-10 weeks old male mice (C57BL/6J; 24-28 g) were purchased from SLAC Laboratory Animal Co., Ltd (Shanghai, China) and were housed in the specific pathogen free (SPF) environment. The mice were bred at 20-25°C at 12h light/dark cycles, and given sterile water and food.
Briefly, animals were randomly (using computer-generated random numbers) divided into two groups (n=13, per group): (1) MI-N.S group, intramyocardial injection of normal saline (N.S); (2) MI-HucMSC group, intramyocardial injection of HucMSCs. Before establishing MI models, HucMSCs were counted and resuspended in N.S at a final concentration of 3*105 cells per mouse. Next, mice were anesthetized with sodium pentobarbital (60mg/kg body weight, intraperitoneal injection), and then intubated and mechanically ventilated using a rodent respirator (Shanghai Alcott Biotechnology Co., Ltd, ALC-V8S). A left thoracotomy at the fourth-fifth intercostal space was performed, and the left coronary artery was ligated. Then, mice were immediately injected 30 μL of 3*105 HucMSCs or N.S by three points into the areas adjacent to the infarcted tissue (10 μL per point) with a 30-gauge needle gas-tight syringe (Hamilton Company). The animal temperature was maintained during surgery by using a heating pad. Finally, the animals were euthanized after 7 or 28 days and tissues were collected for following experiments.
Mice were anesthetized with 2% isoflurane inhalation and analyzed using a Vevo 2100 high-resolution imaging system with a 30-MHz linear transducer (FUJIFILM Visual-Sonics, Inc.) as previously described . We acquired and analyzed the cardiac function of each group through long-axis scans using M-mode images including left ventricular ejection function (LVEF), left ventricular fractional shortening (LVFS), left ventricular end diastolic/systolic volume (LVEDV/LVESV) and left ventricular end diastolic/systolic diameter (LVEDD/LVESD) on day 7 and 28.
Fluorescent labeling of HucMSCs
HucMSCs at P3 were labeled with letivirus containing green fluorescence protein (GFP) at a multiplicity of infection of 10 as previously described . Infection efficiency was confirmed by GFP fluorescent signal viewed under the microscope.
Bioluminescence imaging in vivo
HucMSCs were pre-stained with the near-infrared fluorescent-lipophilic tracer 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR, ThermoFisher, D12731) according to the manufacturer’s instructions, and then 3*105 DiR-labeled HucMSCs were injected in the heart of mice after MI operation. The fluorescence intensity of multiple organs (including heart, lung, liver, kidney and spleen) was analyzed using the IVIS imaging instrument (Xenogen, USA) with the relevant channel.
Polymerase chain reaction (PCR)
Genomic PCR for human Alu-sx repeat sequences in heart tissue was performed as previously reported . The primer of human Alu-sx is: F:5’-GGCGCGGTGGCTCACG-3’, R:5’-TTTTTTGAGACGGAGTCTCGCTC-3’. The amplified products were determined by electrophoresis in 1.5% agarose gel supplemented with ethidium bromide.
Firstly, frozen sections were fixed with 4% PFA and blocked with 1% Bovine Serum Albumin (BSA) at 4°C for 30 min. Then, the sections were incubated with wheat germ agglutinin (WGA, Thermo Fisher, W11261), anti-CD31 (Cell Signaling, 3528S), anti-CD3 (Abcam, ab231775), anti-CD4 (Abcam, ab183685), anti-FoxP3 (Abcam, ab215206), anti-Ki67 (Abcam, ab16667) and human mitochondrion antibodies (Abcam, ab92824), at 4°C overnight to visualize cardiac cells, immune cells and HucMSCs in heart tissue, respectively, while 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) was used to mark cell nuclei. After incubated with above antibodies and washed with phosphate buffered saline (PBS), different fields of each slide were randomly selected and photograph under an inverted fluorescent microscope (Leica DM6000B, Germany).
Real-time quantitative PCR (RT-qPCR)
According to the manufacturer’s instruction, the total RNA was extracted with TRIzol regent (Beyotime, R0016), and then the concentration of total RNA was quantified with NanoDrop2000 (Thermo Fisher Scientific, Inc.). cDNA was obtained by reverse transcription PCR using the PrimerScript RT reagent kit (Takara, RR0036A) at 37°C for 15 min, and 85°C for 5 sec. The mRNA levels were detected by RT-qPCR with a SYBR Green Master Mix kit (Thermo Fisher, 4385617) using an ABI QuantStudio 6 Flex System. The mRNA levels were normalized to the endogenous control GAPDH. The relative gene quantification was calculated using the 2-ΔΔCTmethod. Gene primers were listed in Table S1.
Preparation of single cell and Flow cytometry analysis
Briefly, mice were anesthetized with 1% sodium pentobarbital (60mg/kg, intraperitoneal injection), and the heart was exposed and perfused with pre-cold PBS on day 7 after MI. Then, the heart was cut into 1 mm and put into the C-tube with collagenase II solution (1.5 mg/mL, added 500x DNAse). During broken twice using gentleMACS™ Tissue Dissociators (Miltenyi Biotec, USA), the heart tissue was centrifuged at 37°C for 30 min. After removing debris, the supernatant was filtered (70 μm strainer) to obtain single cell suspension. Cells were stained with the following labeled antibodies: anti-mouse CD45 (Biolegend, Clone I3/2.3), CD19 (Biolegend, Clone 1D3/CD19), CD3 (Biolegend, Clone 17A2), CD4 (Biolegend, Clone GK1.5), F4/80 (Biolegend, Clone BM8), Ly-6G (Biolegend, Clone 1A8). Data were acquired on a FACS Beckman flow cytometer (BD Biosciences, USA), and analysis was performed with Flow Jo software (BD Biosciences, USA).
Cytokine profiling detection
For cytokine protein expression, the tissue samples of infarct and border area in heart of MI murine were collected and weighed. Then we added 500 μL ProcartaPlex Cell Lysis Buffer (EPX-99999-000) per 100 mg tissue. After tissue homogenization and centrifugation, the protein of sample was quantified by Bicinchoninic Acid (BCA) Protein Assay Kit (Bio-Rad, Hercules, CA, USA) and then detected for cytokine profiling (Thermo Fisher, EPX110-20820-901, EPX01A-20614-901, EPX01A-26005-901 and EPX01A-26009-901).
The purification and culture of murine CD4+ T cells from spleen
First, RPMI 1640 complete medium (FBS 5 mL; PS 500 μL; HEPES buffer solution 500 μL; Nonessential Amino Acids 500 μL; Sodium Pyruvate solution 500 μL) and magnetic bead buffer (PBS 48.5 mL; FBS 1 mL; 1mM EDTA 500 μL) were prepared. Next, the spleen was clipped and ground after the mice were anesthetized. Then, primary CD4+ T cells in the spleen were isolated by immunomagnetic bead (Thermo Fisher, 11415D). The density of CD4+ T cells was adjusted by cell count (1*106 cells/per well) and planted to 24-well plate. CD4+ T cells were cultured with RPMI1640 complete medium and stimulated with anti-CD3/CD28 magnetic beads (cells:magnetic beads=5:1, Gibco 00702195).
Enzyme-linked immunosorbent assay (Elisa)
The concentrations of vascular endothelial growth factor (VEGF)-α in the tissue supernatants were measured using Quantikine Elisa kits for VEGF-α (Multi Sciences, EK283/2), according to the manufacturer’s instructions. All results were normalized to the total protein content.
Cell migration assay
Cardiac tissues containing infarct and border zone in MI-N.S and MI-HucMSC groups were collected and lysed and the protein quantified by BCA assay. The concentration of C-C Motif Chemokine Ligand 5 (CCL5) was quantified by Elisa assay (absin, abs520014) according to the manufacturer’s instructions. Polycarbonate membrane transwell inserts (24 well, pore size 5.0 μm) were used for CD4+ T cell migration assay. At the lower chamber, a total volume of 500 μL RPMI1640 containing 20 μg tissue protein were added. At the upper chamber, a total number 5*105 purified CD4+ T cells were added. Sufficient dose CCL5 antibody (R&D system, MAB478) or CCL2 antibody (R&D system, AF-479-NA) was added to the lower chamber for neutralization with information of CCL5/CCL2 concentration determined by Elisa assay (For example, add 0.5 μg/mL of CCL5 antibody per 0.025 μg/mL of CCL5). Sufficient dose (10 ng/mL) C-C Motif Chemokine receptor 5 (CCR5) antagonist (MCE, HY-100261) or CCR2 antagonist (MCE, HY-108323) was added to the upper chamber with purified CD4+ T cells in indicated groups. After co-culturing for 16 h, the CD4+ T cells that had migrated to the lower chamber were collected and counted by handheld automated cell counter (millipore).
The study was performed using GraphPad analysis software (version 8, USA), and data were expressed as mean ± Standard Error of Mean (SEM). Statistical significance of differences between two groups were compared by an unpaired two-tailed Student’s T-test, while multiple comparsions were applied the log-rank test. The survival curve was estimated by the Kaplan-Meier Test. P<0.05 was defined as statistically significant.