Human Tissues
Ethics approval was obtained from the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster. Written consents were signed by recruited subjects after detailed counselling prior to participation of the study. Full thickness endometrial samples were acquired from 22 women with regular menstrual cycles (median age 45.5; range: 41- to 52-years) who underwent total abdominal hysterectomy for benign non-endometrial pathologies (supplementary data Table S1). They had not taken hormonal therapy for three months before surgery. The phase of the menstrual cycle was categorized into proliferative (n = 9) and secretory (n = 13) by experienced histopathologists based on haematoxylin-eosin-stained endometrial sections. Menstrual phase samples were collected by endometrial aspiration from 23 women with regular menstrual cycles and aged from 32 to 43 years attending the infertility clinic on day 2-3 of their menstrual cycle (median age 35; range: 32- to 43-years, supplementary data Table S2).
Single Cell Suspensions of Endometrial Epithelial and Stromal Cells
The isolation procedure of endometrial cells was carried out as described (5). The tissues were minced and digested with PBS containing collagenase type III (0.3 mg/ml, Worthington Biochemical Corporation, NJ, USA) and deoxyribonuclease type I (40 μg/ml, Worthington Biochemical Corporation) for one hour at 37oC. Red blood cells were removed using Ficoll-Paque (GE Healthcare, Uppsala, Sweden) density-gradient centrifugation. Leukocytes were excluded using anti-CD45 antibody-coated Dynabeads (Invitrogen, Waltham, MA, USA). Epithelial cells were separated from the stromal cells using anti-CD326 (EpCAM) antibody-coated microbeads (Miltenyi Biotec Inc., San Diego, CA, USA). Freshly isolated epithelial cells and stromal cells were used for coculture and collection of condition medium as describe below. Some of the spare purified stromal cells (6000-8000 cells/cm2) were plated into 100-mm petri dishes coated with fibronectin (1 mg/ml, Invitrogen) and cultured in growth medium containing 10% FBS (Invitrogen), 1% antibiotics (Invitrogen) and 2 mmol/L glutamine (Invitrogen) in DMEM/F12 (Sigma-Aldrich, St Louis, MA, USA) for 7-14 days in a humidified carbon dioxide incubator at 37oC in 5% CO2. The medium was changed every seven days until the cells reached 90% confluence.
Magnetic bead selection for endometrial mesenchymal stem-like cells
EMSCs were isolated by sequential beading with magnetic beads coated with anti-CD140b and anti-CD146 antibodies as described (5). The stromal cells were first successively incubated with PE-conjugated anti-CD140b antibody (10 μl/106 cells, R&D Systems, Minneapolis, MN, USA) for 45 min at 4oC and then with anti-mouse IgG1 coated microbeads (Miltenyi Biotec Inc.) for 15 min at 4oC before they were loaded onto Miltenyi MS columns with a magnetic field to collect the CD140b+ cells. The stromal CD140b+ population was cultured in fibronectin-coated dishes containing growth medium at 37oC in 5% CO2 for 7-10 days to allow detachment of the microbeads during cell expansion. They were then trypsinized and incubated with anti-CD146 antibody coated microbeads (Miltenyi Biotec Inc.) for 15 min at 4oC. The CD140b+CD146+ cells (eMSCs) were obtained after magnetic column separation.
Coculture
The eMSCs at clonal density (350 cells per well) and the endometrial epithelial or stromal cells (30,000 cells) were seeded onto fibronectin coated 6-well plates and transwell inserts (EMD Millipore, Billerica, MA, USA), respectively and were cocultured. Monoculture (culture of eMSCs without niche cells) served as the control. All conditions were performed in duplicates or triplicates.
Preparation of other cell types
The human oviductal epithelial E6/E7 (OE-E6/E7) cell line (passage 24–26, obtained from Dr CYL Lee) and human foreskin fibroblast (HFF-1) cell line (passage 16–19, CRL-2429, ATCC, Mananas, VA, USA) were also cocultured with eMSCs. The eMSCs were seeded at clonal density (350 cells per well) onto fibronectin coated 6-well plates and OE E6/E7 or HFF-1 cells were seeded at 15,000 cells per insert. New inserts containing OE E6/E7 or HFF-1 cells were replaced on day 7 of culture.
Colony Forming Activity
The number of colony forming units (CFUs) was recorded on day 14 of culture. The colony forming ability was determined by the number of CFUs divided by the number of cells seeded, multiplied by 100 (5).
Flow cytometry
The coexpression of eMSC markers, CD140b and CD146 on endometrial stromal cells after 15 days of culture was analysed using multi-colour flow cytometry as described (16). Endometrial cells were labelled with phycoerythrin (PE)-conjugated antibody against CD140b, (2.5 μg/ml, mouse IgG1, R & D Systems) and fluorescein isothiocyanate (FITC)-conjugated anti-CD146 antibody (1mg/ml, mouse IgG1, Thermo Fisher Scientific, Waltham, MA USA) or isotype matched controls. Flow cytometry analysis was performed using a BD Fortressa (BD Biosciences, San Jose, CA, USA) and the FlowJo software (Tree Star, Ashland, OR, USA) at the Centre for PanorOmic Sciences (CPOS) Imaging and Flow Cytometry Core, The University of Hong Kong.
Preparation of conditioned medium
Conditioned medium (CM) was collected from endometrial cells in the menstrual phase. Freshly isolated epithelial cells or stromal cells (30,000 cells) were cultured in growth medium in 6-well plate for one day then washed with PBS and replaced with 3 ml of growth medium. After 2-days in culture, the CM was collected, filtered sterilised and stored at -80oC until experimentation.
To concentrate the secretory factors from endometrial niche cells, the epithelial or stromal cells were cultured in 1 ml of serum-free DMEM/F-12 medium. The secretory factors in the CM were concentrated (CCM) after 48 hour by centrifugation at 4000g for 20 minutes at 4°C using Amicon ultra-15 centrifugal filter devices (EMD Millipore) with a molecular weight cutoff of 10 kDa. The amount of the concentrated protein derived from one culture well was considered as one unit. Epithelial or stromal CCM (1/3-unit) was added into the growth medium for eMSC culture. The CCM collected from cell free DMEMF-12 medium was used as control.
Western blot analysis
The cellular proteins of eMSCs were extracted with cell lysis buffer (Ambion, Grandisland, NY, USA). The proteins (5 μg) were mixed with 5X SDS loading buffer (60 mM Tris-HCl, pH 6.8, 2% SDS, 0.1% bromophenol blue, 25% glycerol and 14.4 mM β-mercaptoethanol), denatured at 95oC for 10 minutes, subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes (ImmobilonTM-P, Milllipore). The membranes were blocked with 5% skim milk in PBS containing 0.1% Tween-20 for 30 minutes, incubated with primary antibodies at appropriate concentrations (supplementary data, Table S3) overnight at 4°C, and stained with appropriate horseradish peroxidase-conjugated secondary antibodies (supplementary data, Table S3) for one hour at room temperature. The protein bands were visualized by the WesternBright ECL Kit (Advansta, California, USA). The intensities of the protein bands was quantified densitometry and the values were normalized to β-actin using the ImageJ software (US National Institutes of Health, USA).
Quantitative real-time polymerase chain reaction
Total RNA was extracted with the Absolutely RNA RT-PCR microprep kit (Agilent Technologies, Santa Clara, CA, USA). The quality and quantity of the total RNA was checked by spectrophotometry. The RNA was reverse transcribed by the high capacity complementary DNA reverse transcription kit (Roche Applied Science, Basel, Switzerland). Taqman probe for RSPO1 was used (Applied Biosystems, Grand Island, NY, USA) Real-time PCR was performed with a 7500 Real-Time PCR System (Applied Biosystems) using the following parameters: 2 minutes at 50°C, 10 minutes at 95°C, then 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. The results are presented as relative gene expression compared with the internal control 18S using the 2-ΔΔCt method. Determination was made in triplicate from three separate samples.
WNT reporter assay
EMSCs at a density of 20,000 – 50,000 per well were seeded into a 24-well plate. They were co-transfected with 4 μg of either TOPflash or FOPflash vector and 1 μg of pRL-TK (Renilla-TK-luciferase vector, Promega, Madison, WI, USA) as a control using Lipofectamine 2000 (Invitrogen). Cells were subsequently treated with epithelial cell CCM from the menstrual phase (CCM 1/3 unit: growth medium) with or without the neutralization antibodies against RSPO1 (1 μg/ml, Abcam, Cambridge, UK) for 48 hours. Rabbit IgG was the isotype control (Abcam). The cells were lysed and the luciferase activities were measured using a GLOMAXTM 96 microplate luminometer. Firefly luciferase activity was normalized against the Renilla luciferase activity for transfection efficiency. The TOP/FOP ratio was used as a measure of TCF/LEF transcription.
Inhibition of WNT signaling
EMSCs seeded at clonal density were treated with epithelial CCM from the menstrual phase (1/3 unit: growth medium) with or without IWP-2 (Sigma-Aldrich) at 1.25 µM. Growth medium supplemented with dimethyl sulfoxide was used as negative control.
Treatment with neutralization antibodies and recombinant proteins
Neutralization antibody for RSPO1 (1 μg/ml, Abcam) was added to the epithelial CCM from the menstrual phase (1/3-unit: growth medium). Isotype antibody rabbit IgG was used as negative control. Recombinant human WNT3A (12.5, 25, 50 ng/ml, R&D Systems) and RSPO1 (50 ng/ml, R&D Systems) was supplemented to the growth medium of eMSCs seeded at clonal density for 15 days.
Immunofluorescence staining
The unfractionated endometrial stromal cells or eMSCs (8000 – 10,000 cells) were resuspended in growth medium and transferred to slides coated with 3-aminopropyl-triethoxy silane using a Shadon Cytospin Centrifuge (Thermo Electron, Waltham, USA) with centrifugation at 7500 rpm for 10 minutes followed by fixation in 4% paraformaldehyde for 20 minutes. Permeabilization was performed using 0.1% Triton-X 100 for 10 minutes and blocked with the corresponding serum for 30 minutes at room temperature. The slides were then incubated with the primary antibody (supplementary data Table S4) overnight at 4°C, incubated with the secondary Alexa fluor donkey anti-rabbit 568 antibody (Thermo Scientific) for 1 hour at room temperature. The cell nuclei were detected by DAPI (Thermo Scientific). Images were captured with a LSM 700 inverted confocal microscope and a LSM ZEN 2010 software (Carl Zeiss, Munich, Germany) at the Centre for PanorOmic Sciences (CPOS) imaging and Flow Cytometry Core, The University of Hong Kong.
Cytotokine array and ELISA
Cytokine Array C3 (RayBiotech Inc., Norcross, GA, USA) was used to determine the cytokines in coculture experiments. The signal intensities of the cytokines were quantified using the Image J software (NIH Image, National Insistutes of Health, USA). A fold change ≥ 3 after coculture was considered as potential cytokine candidate. The chemokine (C-X-C motif) ligand 1 (CXCL1), CXCL5, Granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-6 (IL-6), monocyte chemoattractant protein 3 (MCP-3) levels serum free CM collected from coculture experiment of eMSCs with endometrial epithelial and stromal cells from the menstrual phase were determined using enzyme-linked immunosorbent assays (ELISA; IL-6, Invitrogen; CXCL1, CXCL5, GM-CSF and MCP-3, R&D Systems) Four candidate cytokine were shortlisted and recombinant CXCL1 (1000 pg/ml; PeproTech, Rocky Hill, NJ, USA), CXCL5 (600 pg/ml; PeproTech), GM-CSF (500 pg/ml, PeproTech) and IL-6 (500 pg/ml, PeproTech) at concentrations found in the coculture condition was added to the growth medium of the eMSCs seeded at clonal density (500 cell/cm2) for 15 days.
Gene silencing
EMSCs were plated in 48-well plates at a density of 8 x 103/ well in OptiMEM (Invitrogen) and the following day transfected with 10 pmol of siRNA directed against LGR5 (ID s16275, Ambion) or random siRNA with scrambled sequence (Ambion) using Lipofectamine RNAiMax transfection reagent (Invitrogen) according to the manufacturer’s instructions. Twenty four hours after transfection, the medium was replaced with OptiMEM. The cells were then assayed using the WNT reporter system as described (9). The knockdown efficiency was assessed by western blotting (supplementary data Fig S1J).
Statistical analysis
Data were analyzed using the GraphPad PRISM software (version 5.00; GraphPad Software Inc., San Diego, CA, USA). Distribution normality was examined using the D’Agostino and Pearson test. Mann-Whitney test was performed to determine statistical significance between two groups. Kruskal-Wallis test followed by Dunn’s post-test were used for multiple group comparison. Data are presented as mean ± SEM. P < 0.05 was considered statistically significant.