Isolation and culture of MenSCs and BMSCs
Menstrual bloods were obtained from five healthy female volunteers with mean age of 25–35 years in the second day of menstruation by sterile Diva cup (Diva International). Isolation and culture of stem cells from MB were previously described by our group . Briefly, the contents of Diva cup along with 2.5 μg/mL fungizone (GIBCO), 100 μg/mL streptomycin, 100 U/mL penicillin (Sigma-Aldrich) and 0.5 mM ethylenediaminetetraacetic acid (EDTA) in phosphate buffered saline (PBS) without Ca2+ or Mg2+ were transferred into the falcon tube. MB derived mononuclear cells were separated by a density gradient centrifugation using Ficoll-Paque (GE Healthcare). MenSCs cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM-F12) medium (GIBCO) with 10% FBS at 37 °C with 5% CO2 and saturated humidity. After one-day, removal of non-adherent cells was performed, and the culture of adherent cells continued until 70–80% confluency. Adherent cells were detached using trypsin (Gibco, UK) and EDTA suspended in PBS.
BMSCs were separated from bone marrow aspirates (5–10 mL) of five healthy female donors aged 18–30 years. The samples were isolated from iliac crests at the Bone Marrow Transplantation Center, Shariati Hospital, Tehran University of Medical Sciences. The isolation procedure of BMSCs was performed using a combination of density gradient centrifugation and plastic adherence as described in our previous study [26, 27]. All experiments were carried out on MenSCs and BMSCs at passages 2–4.
Identification of cultured MenSCs and BMSCs by flow cytometry
Evaluation of the expression of mesenchymal stem cell surface markers (CD73 and CD44), embryonic stem cell surface marker (OCT-4), and hematopoietic cell marker (CD45) was done by flow cytometric analysis as described previously . Cells (105 cells/100 μl) were gently washed in PBS containing 2% FBS and incubated separately with PE-conjugated mouse anti-human CD73 (561014; BD Pharmingen), CD44 (550989; BD Pharmingen), and CD45 (560975; BD Pharmingen) in darkness for at least 40 min at 4 °C. Evaluation of OCT-4 expression was analysed using indirect intracellular flow cytometry. The cells were permeabilized with 0.1% saponin. Then primary rabbit antihuman OCT-4 antibody (ab19857, Abcam) was added for 40 min and then incubated with FITC-conjugated goat anti-rabbit Ig (Sigma-Aldrich) for 30 min. As negative controls, isotype IgG (555748; BD Pharmingen) was used. Afterward, cells were washed twice with PBS-FBS, fixed in 1% formaldehyde solution, and analyzed by a flow cytometer (Partec GmbH).
Pre-treatment of MenSCs and BMSCs with 5-aza and bFGF
MenSCs and BMSCs were cultured and expanded under normoxic condition (O2: 20% CO2: 5%). When cells reached 80% confluency, they were treated with serum-free DMEM consisting 10 μM 5-aza and 10 ng/mL bFGF. After 48 h, CM was collected from monolayer culture of 2 × 106 MenSCs and BMSCs.
Western blot analysis
The expression level of VEGF, HGF, Hypoxia-inducible factor 1-alpha (HIF-1α), Interleukin 1 beta (IL-1β), ANG-1, ANG-2, and Stromal cell-derived factor-1 (SDF-1) in MenSCs and BMSCs derived CM was evaluated before and after pre-treatment with bFGF and 5-aza by western blotting. Western blot analyses were performed as previously described with some modifications . CM was centrifuged at 10 000 rpm for 20 min at 4 °C. Protein concentration was determined by the Bradford protein Quantification kit according to the manufacturer's instructions. The cells lysates were mixed with the equal volume of 2X Laemmli sample buffer. Lysates (15 μg) were separated by SDS-PAGE and subsequently transferred to a 0.2 μm Immune-Blot™ polyvinylidene difluoride (PVDF) membrane (162-017777; Bio-Rad Laboratories). The membranes were blocked with 5% BSA (A-7888; Sigma Aldrich) in 0.1% Tween 20 for 1 h. Then, the membranes were incubated with anti-SDF-1 (ab155090, Abcam), anti- HGF (ab178395, Abcam), anti-VEGF (ab46154, Abcam), anti- Ang-1 (ab183701, Abcam), anti- Ang-2 (ab155106, Abcam), anti-HIF-1α (ab179483, Abcam), anti-IL-1β (ab216995, Abcam), and anti-beta actin-loading control antibodies (ab8227; Abcam) for 1 h at room temperature (RT). Subsequently, membranes were washed with TBST (Tris-buffered saline with 0.1% Tween® 20 Detergent), and incubated with goat anti-rabbit IgG H&L (HRP) (ab6721; Abcam). The membranes were then incubated with enhanced chemiluminescence (ECL) for two min. Protein expression was normalized to β-actin. Densitometry of protein bands was performed using the Gel Analyzer Version 2010a software (NIH, USA), such that, the percentage area under the curve of each band was divided by the percentage area under the curve of its corresponding actin band, and then calculated values were compared among groups.
In vivo modelling
45 male Wistar rats (10–12 weeks old; weight, 300–350 g) were obtained from the animal laboratory of Iran University of Medical Sciences. They were housed in polycarbonate cages inside a well-ventilated room kept on a 12-h light/12-h dark cycle at an average temperature of 24 ± 2 °C, with 50 ± 10% relative humidity, with a standardized regular diet and water ad libitum.
Induction of MI
The animals were anesthetized with ketamine (75 mg/kg) and xylazine (5 mg/kg) intraperitoneally and intubated and ventilated by a rodent ventilator (tidal volume 2–3 mL, respiratory rate 65–70 per min). After a left thoracotomy at the fourth intercostal space; left anterior descending (LAD) artery coronary was permanently ligated (about 2 mm distal from the tip of the left auricle) using a 6-0 prolene suture for induction of myocardial infarction. MI was successfully approved by the development of a pale colour in the myocardial surface distal to the suture and dyskinesia of the anterior wall . After surgery, the ventilator was removed, and the animals monitored until full recovery. Sham-operated rats (n = 8) experienced a similar procedure without coronary artery ligation. Seven days after induction of MI, the surviving animals were narcotized, and their Fractional shortening (FS) and ejection fraction (EF) were evaluated. Then, the rats were randomly divided into three experimented groups (n = 8): (1) MI: 200 μl PBS; (2) bFGF, 5-aza pre-treated MenSCs: 2 × 106 MenSCs suspended in 200 μl MenSCs-CM; (3) bFGF, 5-aza pre-treated BMSCs: 2 × 106 BMSCs suspended in 200 μl BMSCs-CM. Seven days after MI induction, the rats in group 2–3 received their treatments via tail-vein. Postoperative care was preserved utilizing analgesia and hemodynamic monitoring for 48 h.
Echocardiographic evaluation was performed under light anaesthesia by ketamine and xylazine on days 7 and 35 post-surgery. Transthoracic two-dimensional (2D) guided M-mode echocardiography (General Electric-Vingmed Ultrasound, Horten Norway) was done using a 10 MHz electronic linear transducer. Cardiac parameters such as left ventricle internal diameter in diastole (LVIDd) and left ventricle internal diameter in systole (LVIDs) were obtained after 3–5 consecutive heart cycles. FS and EF were calculated according to the formula respectively: [LVIDd − LVIDs)/LVIDd] × 100 and (LVIDd2 − LVIDs2)/LVIDd2 × 100. The ΔFS (% change in FS for each rat) and ΔEF (% change in EF for each rat) were also calculated.
35 days after the beginning of the process, animals were euthanized, and heart tissues were collected and fixed with 10% neutral buffered formalin (NBF). After dehydration and embedding in buffered paraffin, the samples were sectioned at 5 μm thickness, and stained with haematoxylin and eosin (H&E) and Masson’s trichrome. The infarct size was expressed as the percent ratio (%) of the infarct area, divided by the whole left ventricular (LV) area.
Immunohistochemical assay of NF-κB expression
After deparaffinization and rehydration of the slides, the tissue sections were treated with 3% H2O2 (in methanol) for 10 min. After washing with distilled water for 2 min (3X), the slides were placed in citrate buffer (0.01 M, pH = 6) and heated to boiling for 10 min. The slides were allowed to cool at RT. 2% bovine serum albumin mixed in normal sheep serum (Avicenna Research Institute, Tehran, Iran) was used as blocking agent. After that, the slides were incubated with a primary antibody for NF-κB, (ab16502, Abcam, 1:500) overnight at 4 °C. After washing, slides were incubated with secondary antibody (K5007, DAKO) for 1 h at RT. To visualize immunoreactivity, 3, 3’-Diaminobenzidine (DAB) made up with substrate buffer (K5007, DAKO), was added to slides. Finally, the sections were counterstained with Mayer’s haematoxylin (Sigma) for 2 min, dehydrate, and mounted. The percent of NF-κB expression was quantitatively analysed by ImageJ software (ImageJ, NIH, Bethesda, MD, USA). The slides were examined using a microscope (Olympus BX51) connected to a digital camera (Olympus, DP71) by a veterinary anatomic pathologist.
Tracking of engraftment of injected stem cells using immunohistochemistry
Deparaffinized sections were rinsed with Tris-buffered saline (TBS). The antigen retrieval step was done by microwave heating base using EDTA buffer (pH = 9) and then endogenous biotin was blocked with Biotin-Blocking System (X0590, Dako). For blocking, normal mouse serum (Avicenna Research Institute, Tehran, Iran) was used. To tracking of injected stem cells, the sections were incubated with the mouse anti-human anti-mitochondrial antibody (MAB1273B, Merk, 1:150) overnight at 4 °C. The continuation of the process was the same as the method mentioned above.
Evaluation of angiogenesis
The sections were incubated with anti-cluster of differentiation 31 (CD31) primary antibody (orb10314; Biorbyt, 1:100) overnight at 4 °C. After washing, slides were incubated with the secondary antibody (orb688925; Biorbyt, 1:150) for 2 h at 37 °C. Afterward, the slides were washed and incubated with DAPI (Sigma-Aldrich, D9542) for nuclear staining at RT. Vascular density was quantified by the counting of stained capillary structures in 5 randomly high-power fields (HPF) per sample in the infarct border zone.
Blood samples were collected into non-heparinized tubes and centrifuged at 3000 g for 10 min, and the obtained serum was stored at − 80 °C. The amount of Interleukin-6 (IL-6) (R6000B, R&D Systems), tumour necrosis factor-α (TNF-α) (RTA00, R&D Systems), and IL-1β (RLB00, R&D Systems) in serum samples were measured with an enzyme-linked immunosorbent assay (ELISA). In brief, known concentrations of recombinant rat IL-6, TNF-α, or IL-1β and the experimental samples were added and incubated in polystyrene microtiter plates coated with an antibody against the appointed cytokine, followed by incubation with an enzyme-linked polyclonal antibody directed to the cytokine. Next, a substrate solution for the enzyme was added, and the colour development was stopped by adding 2N H2SO4. The absorbance was measured with a microtiter plate spectrophotometer. The amount of IL-6, TNF-α, and IL-1β in each sample was determined from a standard curve generated in each assay and expressed as pictograms per millilitre.
All data were expressed as mean ± SD. For comparing the differences among groups, one-way analysis of variance was used. Statistical analyses were performed by SPSS 20.0 software (IBM Corp., Armonk, NY, http://www.ibm.com). P value < 0.05 was accepted to be a statistically significant difference.