Isolation and culture of human endometrial stem cells

Human endometrial stem cells were obtained with written informed consent from uterine fibroid patients and approved by the Gachon University Institutional Review Board (IRB No: GAIRB2018-134). The endometrial tissue was minced and digested in DMEM containing 10% FBS and 250 U/ml type I collagenase for 5 h at 37 °C in a rotating shaker, according to a previously established procedure [17]. The resulting mixture was filtered through a 40 µm cell strainer to separate stromal-like stem cells from epithelial gland fragments and undigested tissue. Endometrial stem cells were isolated from other cell types using a single-density Percoll layer by centrifugation for 20 min at 1200 g. The isolated cells were washed twice in PBS and cultured in growth media consisting of various growth factors, including IGF, VEGF, EGF, basic FGF, hydrocortisone, ascorbic acid, heparin, and 10% FBS (Gibco BRL) at 37 °C in a humidified atmosphere of 5% CO2 in air. After 3 days, colony-forming cells were isolated using cloning rings (Sigma-Aldrich).

Isolation and culture of mouse uterine tissue-derived stem cells

The isolation of stem cells derived from mouse uterine tissue was approved and conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) (LCDI-2019–0169) of Gachon University. Uterine tissue was minced into small pieces and digested in DMEM containing 10% FBS and 250 U/ml type I collagenase for 5 h at 37 °C. The resulting mixture was filtered through a 40 µm cell strainer, and the isolated cells were cultured in EBM-2 medium (Lonza) with EGM-2 supplements at 37 °C and 5% CO2.

Cell proliferation assay

To determine the anti-proliferative capacity of acetylsalicylic acid, the MTT assay was performed following the manufacturer's protocol. Endometrial stem cells (2 × 104 cells/well) were seeded in 96-well plates and incubated for 24 h. The cells were then treated with increasing concentrations of acetylsalicylic acid for 48 h. The viable cells were measured at a wavelength of 570 nm using a VersaMax microplate reader.

Protein isolation and western blot analysis

We analyzed protein expression levels using western blotting as previously described in our studies [17]. We lysed cells in a buffer containing 50 mM Tris, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP 40, and 0.2 mM PMSF, and measured the protein concentrations of the total cell lysates using bovine serum albumin as a standard. We separated samples containing equal amounts of protein using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred them onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories), and blocked the membranes with 5% skim milk in Tris-buffered saline containing Tween-20 at room temperature. We then incubated the membranes overnight at 4 °C with primary antibodies against MMP-2 (Cell Signaling #4022), MMP-9 (Cell Signaling #13,667), SERPINB2 (Abcam, MA, USA, ab47742), total PI3K (Cell Signaling #4292), phospho-PI3K (Cell Signaling #4228), total Akt (Cell Signaling #4491), phospho-Akt (Cell Signaling #4060), total-ERK1/2 (Cell Signaling #9012), phospho-ERK1/2 (Cell Signaling #9101), total FAK (Santa Cruz, sc-558), phospho-FAK (Santa Cruz, sc-11765), or β-actin (Abcam, ab189073), and subsequently incubated them with polyclonal HRP-conjugated goat anti-mouse IgG (BD Pharmingen, 554,002) or goat anti-rabbit IgG (BD Pharmingen, San Diego, CA, USA, 554,021) secondary antibodies at room temperature for 60 min. Finally, we detected the antigen–antibody complexes using Western blot ECL reagents (GE Healthcare, Bucks, UK).

In vitro cell migration assay

We assessed the impact of acetylsalicylic acid on the migration ability of endometrial stem cells by calculating the ratio of cells that migrated in response to acetylsalicylic acid treatment versus the number of cells that migrated spontaneously. To track cell migration, we plated cells at a density of 1 × 105 cells/well in 200 μL of culture medium in the upper chambers of permeable Transwell supports (Corning Inc., Corning, NY, USA). These Transwell chambers had 8.0 μm pores in 6.5-mm diameter polycarbonate membranes and were arranged in a 24-well plate format. After incubation, non-migrated cells on the upper surface of each membrane were removed by scrubbing with laboratory paper. Migrated cells on the lower surface of each membrane were then fixed with 4% paraformaldehyde for 5 min and stained with hematoxylin for 15 min. Finally, we counted the number of migrated cells in three randomly selected fields of each well using a light microscope at 50X magnification. The difference between the groups was expressed as a fold change.

Real-time PCR analysis

Total RNA was extracted from human or mouse endometrial stem cells using commercial TRIzol® reagent (Invitrogen Life Technologies, CA, USA) according to the manufacturer’s recommended instructions. RNA purity was estimated by measuring the ratio of absorbance at 260 nm and 280 nm. The first-strand cDNA was synthesized using SuperScript II Reverse Transcriptase (Invitrogen Life Technologies, CA, USA) with 1 μg of total RNA. The first-strand cDNA was synthesized using Express SYBR-Green qPCR Supermix (BioPrince, Seoul, South Korea). qPCR was performed using a QIAGEN's real-time PCR cycler, the Rotor-Gene Q. The relative mRNA expression levels of the target genes were calculated as fold changes using the ΔΔCT method. The sequences of the PCR primers are listed in Table 1.

Table 1 Primer sequences for quantitative RT-PCRIngenuity pathway analysis

The Ingenuity Pathway Analysis (IPA) version 2.0 software (Ingenuity Systems, Redwood City, CA) was used to analyze the genes related to SERPINB2. Genes that were differentially expressed between proliferative and non-proliferative cells (t-test, P < 0.005) were subjected to analysis of EGFR (GSE21618), IGFBP6 (GSE47856), M-CSF (GSE45630), PDGFRB (GSE116237), SCFR (GSE46045), or TGFß2 (GSE48990)-related genes. Fisher's exact test (P value) was used to measure the significance of each molecule, identifying differentially expressed genes from the microarray data that overlapped with known regulated genes. The activation score (z score) was used to indicate the predicted molecule status by comparing the observed differential regulation of genes ("up" or "down") in the microarray data relative to the literature-derived regulation direction, which can either activate or inhibit.

Flow cytometry

Flow cytometry analysis and cell sorting were conducted using FACS Calibur and FACS Aria machines (Becton Dickinson, Palo Alto, CA), respectively. Flow cytometry data were analyzed with FlowJo software (Tree Star, Ashland, OR). The antibodies used for detection included PE-conjugated CD34 (MACS; Miltenyi Biotech, 30–081-002, dilution 1/40), CD44 (MACS; Miltenyi Biotech, 130–095-180, dilution 1/40), CD45 (MACS; Miltenyi Biotech, 130–080-201, dilution 1/40), CD73 (MACS; Miltenyi Biotech, 130–095-182, dilution 1/40), CD105 (MACS; Miltenyi Biotech, 130–094-941, dilution 1/40), CD140b (MACS; Miltenyi Biotech, 130–105-279, dilution 1/40), and W5C5 (MACS; Miltenyi Biotech, 130–111-641, dilution 1/40). FACS gates were set using either an isotype antibody or a secondary antibody.

Analysis of Gene Expression Omnibus (GEO) database

Gene Expression Omnibus (GEO) is an open-access database repository that stores high-throughput gene expression data generated by genome hybridization arrays, chip sequencing, and DNA microarrays [18, 19]. Researchers can upload their experimental results in four categories: experimental designs, sample, platform, and raw data. Within each dataset, clinical or experimental samples are further classified into various experimental subgroups based on treatment, physiologic condition, and disease state. This categorized biological data is presented as a "GEO profile," which includes the dataset title, gene annotation, a chart showing the expression levels and rank for each gene across the samples [20]. To analyze the expression profiles of EGFR, IGFBP6, M-CSF, PDGFRB, SCFR, or TGFß2 in response to various acetylsalicylic acid treatment conditions, we followed previously established procedures [20].

Adipocyte differentiation

Human or mouse endometrial stem cells were cultured in low-glucose DMEM supplemented with 500 µM methylxanthine, 5 µg/mL insulin, and 10% FBS for 3 weeks with medium change twice per week. The formation of lipid droplets was confirmed by oil red O staining, and their abundance was measured by calculating the absorbance at 500 nm.

Osteoblast differentiation

Endometrial stem cells derived from either human or mouse were cultured in high-glucose DMEM supplemented with 0.1 µM dexamethasone, 10 mM β-glycerophosphate, 50 µM ascorbate, and 10% FBS for a period of three weeks with medium replacement twice per week. After differentiation, cells were stained with alizarin red S to detect the formation of new bone matrix. To quantify the presence of alizarin red S in the samples, the optical density (OD) of the solution was measured at 570 nm.

SERPINB2 knockdown

Bioneer (Daejeon, South Korea) provided small hairpin RNA (shRNA: accession No. NM_002575) targeting SERPINB2 and scrambled shRNA (shCon). To achieve efficient SERPINB2 transfection, reverse transfection was conducted using Lipofectamine 2000 (Invitrogen) as per the manufacturer's instructions. Specifically, shRNA targeting SERPINB2 (3 μg/ml) was mixed with 3 μl transfection reagent lipofectamine 2000 in Gibco opti-MEM media without FBS and antibiotics. Five hours before transfection, opti-MEM was replaced with fresh EGM-2 medium containing 10% FBS. The most effective SERPINB2 shRNA, as determined by qRT-PCR analysis, was selected from three shRNA designed from the target sequence.

Growth factor antibody array

The manufacturer's protocol (Abnova AA0089) was followed to perform the assay. In brief, protein samples treated with acetylsalicylic acid or vehicle were incubated with antibody membranes overnight at 4 °C. After washing with wash buffer for 3 times, biotin-conjugated anti-cytokine antibodies were incubated with the membranes overnight at 4 °C. The membranes were then washed 3 times and incubated with HRP-conjugated streptavidin. Detection of signals of the growth factors that were spotted on the nitrocellulose membrane was performed using chemiluminescence.

Evaluation of the effects of acetylsalicylic acid treatment in animal model

Even though it does not entirely depict the physiological features of the human body, the outcomes achieved in vitro were validated through the use of mice, which are commonly utilized as in vivo models for evaluating efficacy. Animal experiments were conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) (LCDI-2019–0169) of the Gachon University, and all protocols were approved. All experiments were designed and reported in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. The animals were housed in standard cages with ad libitum access to food (standard chow diet) and water. The animal room was maintained on a 12-h light/dark cycle at a constant temperature of 22 °C. The female mice (C57BL/6) were blindly and randomly divided into two groups (n = 5 per group sufficient to determine differences by treatment): control and acetylsalicylic acid treatment (50 mg/kg for 7 consecutive days intravenously). All animals were involved in data analyzing. After anesthesia and exsanguination by cardiac puncture, stem cells were isolated from uterine and adipose tissues. The isolated stem cells from endometrium, adipose tissue, or bone marrow were then cultured and expanded in vitro with continuous exposure to acetylsalicylic acid (2.5 mM) to simulate the physiological conditions of acetylsalicylic acid exposure in vivo. Then, the effects of aspirin on the self-renewal, migration, pluripotency, and differentiation capacity of stem cells in vivo were evaluated.

Statistical analysis

The experimental data were presented as mean ± standard deviation (SD) and were obtained from a minimum of three independent experiments. Statistical analysis among the experimental groups was performed using GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, US) with one-way ANOVA. Significance was set at p < 0.05.