2.1. Mononuclear cells isolation and expansion protocol
Bone marrow samples of healthy volunteers, ranging from 3 to 10 years old, referred to children hospital, an affiliated hospital to Tabriz University of Medical Sciences, were enrolled in the present study. Informed consent was obtained from candidates. Briefly, bone marrow aspirates (2 ml) were used for the isolation of MNCs. In order to inhibit the coagulation, 5,000 IU/mL heparin was used. Blood samples were diluted with PBS (1:1 v/v) and overlaid on the same volume of Ficoll-Hypaque solution (Cat No: GE17-1440-02; Sigma-Aldrich). The samples were centrifuged at 400 g for 25 minutes at 4 °C. MNCs at the interface fraction between the plasma and the Ficoll solution was carefully collected, washed with PBS and re-suspended in DMEM/LG (Cat No: 31600083; Gibco; USA) culture medium. The media were supplemented with %10 FBS (Cat No: 10270; Invitrogen). The medium was replaced every 3–4 days. By using 0.25% Trypsin-EDTA (Cat No: 25200056; Gibco; USA) solution, cells were detached.
2.2. Enrichment of CD146+ cells using Magnetic Activated Cell Sorting
In the current study, we aimed to isolate CD146+ cells for different analyses. For this purpose, expanded bone marrow MNCs were detached using the enzymatic solution and subjected to MACS. In short, the MNCs were blocked by using 1% bovine serum albumin for 20–30 minutes and incubated with mouse anti-human CD146 microbead (Order no: 130-093-596; Miltenyi Biotec; Germany) for 30 minutes at 4˚C. The cell suspensions were passed over the MACS LS column (Order no: 130-042-401; Miltenyi Biotec).
2.3. Cell survival assay
This study aimed to evaluate the effect of autophagy modulation on the differentiation capacity of CD146+ cells toward different lineages. In this regard, we performed MTT assay to select the maximum dose of autophagy blocker, HCQ, with the lowest toxic effect on CD146+ cells. In this regard, CD146+ cells were plated (2 × 104/well) in each well of 96-well plates (SPL). Cells were treated with different concentrations of HCQ (Cat No: H0915; Sigma-Aldrich) including 2.5, 5, 10, 15 and 20 µM for 72 hours [19]. Thereafter, 30 µl MTT solution was added to each well and incubated at 37˚C for 2 hours followed by the addition of 200 µl dimethyl sulfoxide (Merck; Germany). The optical density was read at 620 nm by using a microplate reader (BioTek). The cell survival rate was expressed as a percentage relative to the non-treated control CD146+ cells. In order to stimulate autophagy, CD146+ cells were treated with 50 mM Met (As a gift from Osveh pharmaceutical Inc., Tehran, Iran) [20].
2.4. LysoTracker assay
To assess the inhibitory effect of HCQ on the late stage of autophagy, we performed LysoTracker assay. To this end, MNCs were seeded at a density of 104 cells per well in 8-well Chambered Cell Culture Slide (SPL) and incubated at 37 °C with 5% CO2 and 95% relative humidity. After 24 hours, cells were treated with 15 and 20 µM HCQ for 72 hours. After completion of autophagy modulation, cells were washed with cold PBS, and 50 nM LysoTracker Green (Cat No: L7526; Sigma-Aldrich) was added to each well. Cells were allowed to maintain for 30 minutes. After three times washing with PBS, cells were stained with 1 µg/ml DAPI (Sigma-Aldrich) solution 30 seconds in order to stain background. The cells harboring intracellular vacuoles were visualized by using immunofluorescence microscopy (Model: BX41; Olympus).
2.5. Cell differentiation and autophagy modulation
In this study, we explored the effect of autophagy modulation on the differentiation potency of CD146+ cells in vitro. Purified CD146+ cells were cultured in the endothelium (Cat No: C-22111, Promocell, Germany), pericyte (Cat No: C-28040, Promocell, Germany) and cardiomyocyte (Cat No: 05010, STEMCELL, USA) differentiation media. The differentiation media were supplemented with 2% FBS and 1% Pen-Strep solutions and cells were maintained for 7 days. On day 4, autophagy was blocked/stimulated using 15 µM HCQ and 50 µM Met (Cat No: Osveh Pharmaceutical Inc., Iran) as previously described (Fig. 1A) [20].
2.6. Western blotting
Western blot analysis was done to evaluate and confirm the modulation of autophagy proteins in PC-, CM- and EC-like cells after the modulation of autophagy. On day 7, cells were collected and lysed in ice-cold cell lysis buffer solution (NaCl, NP-40, and Tris–HCl) enriched with cocktail enzyme inhibitors. Then, samples were sonicated at a resonance of 50 Hz for 2 minutes. The homogenates were centrifuged at 14,000 g for 20 minutes. By using the Picodrop spectrophotometer system (Model NO: PICOPET01; Serial NO: 000212/1), total protein content was determined in the supernatant. In this study, 100 µg proteins from each sample were resolved by 12% SDS polyacrylamide gel electrophoresis and transferred to PVDF membrane. The membranes were incubated in antibody solution (LC3: Cat No: ab51520, Abcam; P62: Cat No: SC-10117; Santa Cruz Biotechnology, Inc.; Beclin-1: Cat No: SC-48341; Santa Cruz Biotechnology, Inc.; α-actinin-2: Cat No: SC-130928; Santa Cruz Biotechnology, Inc.; α-SMA: Cat No: SC-53015; Santa Cruz Biotechnology, Inc.; vWF: Cat No: SC-365712; Santa Cruz Biotechnology, Inc.; ERK 1/2: Cat No: SC-292838; Santa Cruz Biotechnology, Inc.; pERK 1/2: Cat No: SC-16981-R; Santa Cruz Biotechnology, Inc.) at 4 °C overnight. Then, the membranes were incubated with secondary HRP-conjugated anti-IgG antibody (Cat No: sc-2357; Santa Cruz Biotechnology, Inc.) for 1 hour at room temperature. The immunoreactive blots were detected using the ECL plus solution (BioRad). Immuno-reactive bands were analyzed with ImageJ software (ver.1.44p; NIH). GAPDH (Cat No: sc-32233; Santa Cruz Biotechnology, Inc.) was used as an internal control group. This experiment was performed in triplicate.
2.7. NO assay
The level of NO, either intracellular or released content, was measured in differentiating cells after autophagy stimulation or inhibition using the Griess method. In this method, NO is converted to nitrite and nitrite produces nitrous acid in an acidic state. In the next step, the addition of sulfanilamide to a solution containing nitrous acid forms a diazonium salt and in the presence of n-(1-naphthyl) ethylenediamine dihydrochloride (C12H16Cl2N2) generates an azo dye. Briefly, an initial number of 1 × 104 cells/well was seeded in each well of 96-well plates (SPL). 72 hours after the addition of autophagy blocker/stimulator under differentiation condition, 200 µl supernatant of each sample was mixed with 20 µl Griess A and maintained for 10 minutes followed by the addition of 20 µl of Griess B solution. After 2 minutes, the optical density of each sample was read at 450 nm using a microplate reader (BioTek) and levels of NO calculated based on sodium nitrite standard curve. We also monitored the intracellular level of NO in addition to released content. Levels of NO were expressed as nM.
2.8. Measuring fatty acid composition by GC
The lipid profile was analyzed by the GC technique as previously described [21]. The cellular lipid was extracted by using the Bligh-Dyer method and further esterified with methanol during acetyl chloride catalysis [22]. Methyl esters were introduced to the analyzing machine in order to determine the fatty acid composition. The fatty acid methyl ester derivatives were separated on a Teknokroma TR CN100 column (60 × 0.25 mm) using a Buck Scientific gas chromatograph (Model 610; SRI Instruments, Torrance, USA). Tridecanoic acid (13:0) was exploited as the internal control. Peak retention times in each group were compared to the peak of standards.
2.9. Measuring endothelial RTKs and pro-inflammatory cytokines
To determine the levels of inflammatory cytokines and RTKs during CD146+ differentiation into different lineages, we measured the levels of IL-6 (Cat No: DY206; R&D) and TNF-α (Cat No: DY210; R&D) in the supernatants by using ELISA according to manufacturer's recommendation. The angiogenic status of cells was further assessed by monitoring RTKs protein levels such as VEGFR-1 (Cat No: ab32152; Abcam), VEGFR-2 (Cat No: ab39256; Abcam), Tie-1 (Cat No: ab27851; Abcam), Tie-2 (Cat No: ab24859; Abcam) and Ang-1 (Cat No: 130-06; Peprotech) by ELISA assay provided by our group [23]. The final absorbance was recorded at 450 nm using a microplate reader (BioTek; USA). The concentration of each factor either in autophagy stimulated/inhibited and groups is calculated based on comparison to corresponding standard curves.
2.10. Assessing paracrine potency of CD146+ cells under the modulation of autophagy
To this end, we monitored AChE touted as an exosome marker protein. In brief, supernatants were discarded after the completion of the experimental procedure and replaced by incubation with culture medium containing 1% exosome-free FBS (Invitrogen) for 48 hours. Thereafter, 20 µl culture medium was mixed with 500 µl buffer solution containing pyrophosphate (75 mM) and potassium hexacyanoferrate (2 mM) (Cat No: BXC0801; biorexfars) for 5 minutes at RT. Finally, 100 µl s-butyrylthiocholine iodide was added to each sample and absorbance read at 405 nm during three different intervals. Finally, the choline esterase activity was calculated by the following formula; Activity (U/l) = ∆Abs/min × 65800
2.11. RT-PCR assay
After 7-day incubation with differentiation media and autophagy modulation, the total RNAs were isolated from each sample. To this end, cells were trypsinized and were washed with PBS. Then, 1 ml Ambion TRIzol buffer (Cat No: 15596-026; Invitrogen; USA) was added to cell pellets and agitated rigidly to homogenize the samples. Next, 200 µl chloroform (Merck; Darmstadt; Germany) was added and samples centrifuged at 4 °C at 12000 rpm for 20 minutes. Next, the equal volume of the upper phase containing RNA was mixed with isopropanol (Sigma-Aldrich) and incubated at 4 °C for 15 minutes. After centrifugation, the supernatant was discarded and the pellet was dissolved in 1 ml ethanol (75% v/v) solution. Finally, the total RNA was dissolved in 50 µl DEPC treated water and samples' concentration determined by a Nanodrop system (Thermo Scientific). To exclude a possible genomic DNA contamination, RNA solutions were incubated with the DNase1 kit (Cat No: en0521; FermenTaz). Before real-time PCR analysis, purified RNAs were reversely transcribed using a cDNA synthesis kit (Cat No: YT4500). The qRT-PCR reaction was performed by using 1 µl cDNA, 5 µl SYBR premix Ex Taq kit (Cat No: RR820L; TaKaRa), 0.4 µl candidate gene primers and 3.2 µl DEPC water. The qRT-PCR was performed by a Rotor-Gene Corbett System 6000. The raw data was analyzed by a convenient Pfaffl method with normalization to housekeeping gene β2-microglobulin (β2M). The experiment was conducted in triplicate. The sequences of all primers for human VE-cadherin, cTnI, α-SMA, and TPBG were outlined in Table 1.
Table 1
The list of primers designed by Perl Primer software
Gene | Forward | Reverse | Tm (°C) |
α-SMA | 5'-GTCCACCGCAAATGCTTCTA-3' | 5'-AAACACATAGGTAACGAGTCAG-3' | 57 |
VE-cadherin | 5'-ACCCAAGATGTGGCCTTTAG-3' | 5'-GTGACAACAGCGAGGTGTAA-3' | 60 |
Troponin I | 5'-TTTGACCTTCGAGGCAAGTTT-3' | 5'-CCCGGTTTTCCTTCTCGGTG-3' | 57 |
β2M | 5'-AGGCTATCCAGCGTACTCC-3' | 5'-ATGTCGGATGGATGAAACCC-3' | 58 |
CD63 | 5'-TCCTGAGTCAGACCATAATCC-3' | 5'-GATGGCAAACGTGATCATAAG-3' | 63 |
ALIX | 5'-CTGGAAGGATGCTTTCGATAAAGG-3' | 5'-AGGCTGCACAATTGAACAACAC-3' | 63 |
Rab11 | 5'-CCTCAGCCTCTACGAAGCAAA-3' | 5'-CCGGAAGTTGATCTCCTCCTG-3' | 59 |
Rab27a | 5'-AGAGGAGGAAGCCATAGCAC-3' | 5'-CATGACCATTTGATCGCACCAC-3' | 59 |
Rab27b | 5'-GGAACTGGCTGACAAATATGG-3' | 5'-CAGTATCAGGGATTTGTGTCTT-3' | 59 |
2.12. Statistical analysis
In this study, data are expressed as mean ± SD. To find statistically significant differences between the groups, we performed a One-Way ANOVA test. P < 0.05 was considered statistically significant. Three sets of experiments were at least performed for different analyses unless mentioned.