Cell Lines and Culture
Human trophoblast BeWo cells and their culture medium were purchased from ATCC (Manassas, VA, USA). BeWo cells were cultured in F-12k medium (ATCC) supplemented with 100 U/mL of penicillin (Sigma-Aldrich, St. Louis, MO, USA), 100 μg/mL of streptomycin (Sigma-Aldrich), and 10% fetal bovine serum (FBS) (Gibco by Thermo Fisher Scientific, Waltham, MA, USA). Human fetal stem cells (FSCs) were established using a known method from amniotic fluid obtained from the healthy pregnant woman at 10 weeks of gestation who performed amniocentesis with written informed consent for utilization of tissues and all experimental procedures approved by the Institutional Bioethics Committee of Stemmedicare Ltd. (SIRB-2020-AUG-01). Human hematopoietic stem cells (HSCs) were purchased from ATCC and were maintained in Hank’s Balanced Salt Solution (HSBS) medium (ATCC) supplemented with 100 U/mL of penicillin, 100 μg/mL of streptomycin, and 10% heat inactivated FBS. Human amniotic mesenchymal stem cells (hAM-MSCs) and human umbilical cord blood mesenchymal stem cells (UCB-MSCs) were purchased from ScienCell (Carlsbad, CA, USA). Human amniotic fluid-derived mesenchymal stem cells (hAF-MSCs) were established as described in the previous study (28). These MSCs were maintained in DMEM medium (Welgene, Republic of Korea) supplemented with 100 U/mL of penicillin, 100 μg/mL of streptomycin, and 10% heat-inactivated FBS.
In vivo-like temperature change, pH, and circulation conditions
An in vivo-like culture condition for inducing immune-tolerized cell lines was applied using temperature change, pH, and circulation conditions established in the previous study (28). In summary, temperature change between 36.0 ℃ and 37.0 ℃ with a five or six-day cycle and an acidic pH of 6.2 to 6.8 by HA-based matrix were applied to induce the secretion of various pregnancy-related hormones as shown in Fig.2C. Simultaneously, a circulation condition with a 24-hr cycle, as shown in Fig.2D, was applied to facilitate the signal transduction between the culture matrix and immune-tolerized cells to promote autocrine of various soluble factors.
Establishment of co-culture system of HSCs and UCB-MSCs
An indirect co-culture system of HSCs and UCB-MSCs was established using a multi-dish with polycarbonate membrane insert (Thermo Fisher Scientific). HSCs were seeded at a density of 2 x 105 / cm2 in a 6-well plate, and UCB-MSCs were seeded at a density of 2 x 104 / cm2 in a 0.4 μm pore insert (Nunc). They were cultured in DMEM serum-free medium in a 1% O2 and 5% CO2 humidified atmosphere at 37°C for 120 hrs to obtain the conditioned medium to isolate EVs later.
EVs Preparation
Multi-stage Filtration. To increase the efficiency of the subsequent stage filtration and recovery a high yield of EVs, multi-stage filtration method was applied, established in the previous study (28). In summary, the cell culture supernatant, centrifuged at 1,500 RPM for 5 min, was filtered with a 0.8-μm filter to remove cell debris and apoptotic bodies completely, and the supernatant was filtered with a 0.45-μm filter twice. To obtain small EVs, the supernatant was filtered with a 0.22-μm filter in the same manner as above.
AF/AM-MSCCO-EVs. The conditioned medium obtained from serum-free co-cultivation of hAM-MSCs and hAF-MSCs in multi-dish plate, as described in the previous study (28), was centrifuged at 1,500 RPM for 5 min at the room temperature, and the supernatant was filtered with a 0.45-mm filter after multi-stage filtration to obtain AF/AM-MSCCO-EVs.
HSC/UCB-MSCCO-EVs. The conditioned medium obtained from serum-free co-cultivation of HSCs and UCB-MSCs in multi-dish plate was centrifuged at 1,500 rpm for 5 minutes at the room temperature, and the supernatant was filtered with a 0.22-μm filter after multi-stage filtration to obtain HSC/UCB-MSCCO-EVs.
itTBC-EVs. BeWo cells with both syncytiotrophoblast (STB) and EVT phenotypes were sub-cultured in HA-Matrix 3-5 times to establish immune-tolerized trophoblast cells (itTBCs). The conditioned medium, obtained from serum-free cultivation of itTBCs with a seeding density of 2.0 x 104 / cm2 in HA-Matrix containing AF/AM-MSCCO-EVs with in vivo-like temperature change, pH, and circulation conditions, was centrifuged at 1,500 RPM for 5 minutes at the room temperature, and the supernatant was filtered with a 0.45-μm filter after multi-stage filtration to obtain itTBC-EVs.
NAbs-FSC-EVs. The conditioned medium, obtained from serum-free cultivation of NAb-secreting FSCs (NAbs-FSCs) with a seeding density of 2.0 x 104 / cm2 alone without any matrix after washing with PBS several times to remove any contaminant during the cultivation on HA-matrix containing itTBC-EVs and HSC/UCB-MSCCO-EVs with in vivo-like temperature change, pH, and circulation conditions, was centrifuged at 1,500 RPM for 5 minutes at the room temperature, and the supernatant was filtered with a 0.22-μm filter after multi-stage filtration to obtain NAbs-FSC-EVs.
NTA measurement of EVs with Nanosight NS300
The concentration and size of EVs were analyzed by NTA using a NanoSight NS300 with a Blue 488 nm laser (Malvern Panalytical, Malvern, UK). All samples were diluted in PBS to reach concentrations inside the precision range of the NTA machine (2 × 108 to 10 × 108 particles/ml). EVs were measured at camera level 14 (camera shutter speed: 21.48 shutters / ms, slider gain: 366). After capture, the videos have been analyzed using the in-build NanoSight Software NTA 3.4 Build 3.4.003 with a detection threshold 5.
Flow Cytometry
The expressions of Igs (IgG), NAbs (IgM and IgG3), stem cell and EV-specific markers, and HLA-G proteins in FSCs and FSCs-derived EVs were quantified by flow cytometry. FSCs were harvested and stained with anti-Human CD73 APC (Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA), anti-Human CD90 APC (Invitrogen), anti-Human CD105 APC (Invitrogen), anti-Human SSEA-4 AF488 (Invitrogen), anti-HLA-G 87G APC (BioLegend, San Diego, CA, USA), anti-HLA-G 2A12 FITC (Invitrogen), anti-Human IgG Fc AF488 (BioLegend), anti-Human IgG3 Hinge AF555 (SouthernBiotech, Birmingham, AL, USA), anti-Human IgG3 AF488 (Novus Biologicals, Centennial, CO, USA), and anti-Human IgM AF647 (BioLegend) antibodies for 30 min at room temperature. Intracellular staining of FSCs were performed using Intracellular Fixation & Permeabilization Buffer set (Invitrogen) according to the manufacturer’s instruction.
The EVs were stained with anti-Human CD9 FITC (Invitrogen), anti-Human CD63 PE (Invitrogen), and CD81 APC (Invitrogen) antibodies for 30 min at room temperature. Intracellular staining of EVs were performed after EV isolation using Exosome Isolation and RNA Purification Kit (System Biosciences, Palo Alto, CA, USA) according to the manufacturer’s instruction. Isolated EVs were treated with permeabilizing solution for 1 hour at room temperature and stained with anti-HLA-G 2A12 FITC, anti-Human IgG Fc AF488, anti-Human IgG3 AF488, and anti-Human IgM AF647 antibodies for 30 min at room temperature. Then, stained EVs were isolated again using Exosome Isolation and RNA Purification Kit and analyzed through flow cytometry using various sizes of reference beads (Thermo Fisher Scientific).
The flow cytometry analysis of FSCs and their EVs were performed using Novocyte Quanteon (Agilent Technologies, Santa Clara, CA, USA), and the data were analyzed by Novoexpress software ver. 1.43.
Enzyme-linked immunosorbent assay (ELISA)
The concentration of sHLA-G (shedding HLA-G1 and HLA-G5) was detected by Human HLA-G ELISA Kit (LSBio, Seattle, WA, USA) using MEM-G/9 antibody according to the manufacturer's instruction. The concentration of soluble HLA-G isoforms (HLA-G5 and HLA-G6) was detected by soluble HLA-G ELISA Kit (MyBioSource, San Diego, CA, USA) using 5A6G7 antibody according to the manufacture's instruction.
The concentrations of IgG3 and IgM were analyzed using Human IgG3 ELISA Kit (MyBioSource) and Human IgM ELISA Kit (Abnova, Taipei, Taiwan) according to the manufacturer’s instructions, respectively.
Protein Antibody Microarray
The immunoglobulin and protein profiles in EVs secreted from FSCs were analyzed using SET100 Signaling Explorer Antibody Array (Full Moon Biosystems, Sunnyvale, CA, USA) and Human L493 Array (RayBiotech, Peachtree Corners, GA, USA) according to the manufacturer's instructions.
Cytokine Array Analysis of EVs
The concentrations of human growths and cytokines in EVs were analyzed by Quantibody Human Cytokine Array 1000 (RayBiotech) according to the manufacturer's instruction.
Protein absolute quantification of EVs
ESI-Q-TOF MS/MS analysis is performed on pre-treated EV sample according to the manufacturer’s instructions using Synapt G2-Si HDMS (Waters, Milford, MA, USA) equipment. Protein identification was performed using the human database (version 3.87) of the IPI (International Protein Index). Protein absolute quantification was carried out based on the standard BSA mass value information (SwissProt) according to the manufacturer's instructions and the method suggested by Silva J. et al. (60).
Characterization of EVs with ExoViewTM R100
Co-expressions of CD9, CD61, CD81, syntenin, IgG3, and IgM in EVs were analyzed using Tetraspanin Custom Kit with ExoViewTM R100 (NanoView Biosciences, Boston, MA, USA) according to the manufacture’s instruction of Cargo and Surface Membrane Immuno-Fluorescence Staining. Labelling Abs that consist of anti-syntenin Alexa-555, anti-human IgG3 Alexa Fluor 488 (Novus Biologicals), and anti-human IgM Alexa Fluor 647 Abs (BioLegend), were used. The data were then analyzed using ExoViewer Analyzer 3.0 with sizing thresholds set to 50 to 200 nm diameter.
Statistical analysis
Statistical analyses were performed using the Student’s t-test for the comparison of means in more than two groups. Data presented are mean±S.D. and P<0.05 was considered statistically significant.