Edited by: Antonio Salgado, University of Minho, Portugal
Reviewed by: Daniela Palacios, Santa Lucia Foundation (IRCCS), Italy; Belém Sampaio-Marques, University of Minho, Portugal
†These authors have contributed equally to this work
This article was submitted to Stem Cell Research, a section of the journal Frontiers in Cell and Developmental Biology
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Human amnion-derived mesenchymal stem cells (AD-MSCs) have been reported as a promising effective treatment to repair tissue. Trophoblast dysfunction during pregnancy is significantly involved in the pathogenesis of preeclampsia (PE). To understand how AD-MSCs regulated trophoblast function, we treated trophoblasts with AD-MSC-derived exosomes under hypoxic conditions. The treatment markedly enhanced the trophoblast proliferation and autophagy. Furthermore, significant decrease of EZH2 levels and inactivation of mTOR signaling were observed in AD-MSC exosomes-treated trophoblasts. Consistent with these findings, overexpression of EZH2 activated the mTOR signaling in trophoblasts, and reduced the autophagy and survival of trophoblasts, even in the presence of AD-MSC-derived exosomes. In addition, EZH2 inhibition exhibited the same trophoblast autophagy-promoting effect as induced by AD-MSC-derived exosomes, also accompanied by the inactivation of mTOR signaling. Importantly, when EZH2 was overexpressed in trophoblasts treated with PQR620, a specific mTOR signaling inhibitor, the autophagy and proliferation in trophoblasts were decreased. Studies on human placental explants also confirmed our findings by showing that the expression levels of EZH2 and mTOR were decreased while the autophagy-associated protein level was increased by AD-MSC-derived exosome treatment. In summary, our results suggest that EZH2-dependent mTOR signaling inactivation mediated by AD-MSC-derived exosomes is a prerequisite for autophagy augmentation in hypoxic trophoblasts.
Preeclampsia (PE) is a hypertensive disorder during pregnancy, and is associated with substantial maternal and perinatal complications (
Autophagy is a mechanism that maintains homeostasis through degradation of malfunctioned organelles, redundant molecules and invasive pathogens by lysosomes (
EZH2 is a bona fide histone methyltransferase, methylating histone 3 at lysine 27 (H3K27) and leading to a selective suppression of transcription by changing the chromatin conformation (
Mesenchymal stem cells (MSCs) are versatile cells that can be differentiated into various specialized cells including osteoblasts, scleroblasts, chondrocytes, and adipocytes (
In this study, the effects of AD-MSC-derived exosomes on the proliferative capacities of the trophoblast cell lines JEG-3 and HTR-8 were studied. In addition to increasing the proliferative capacities, AD-MSC-derived exosomes significantly enhanced autophagy in the trophoblasts under hypoxic conditions. The transcriptome analysis showed considerable downregulation of the enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) and mTOR signaling pathway in trophoblasts treated with treated with AD-MSC-derived exosomes; mTOR was putatively recognized as an upstream inhibitor of autophagy under hypoxic conditions.
Placentas were obtained from full-term births after a cesarean section (
The JEG-3 and HTR-8 cells obtained from the Type Culture Collection of China Centre were subjected to culture and then used for experiments. DMEM/F12 containing 10% FBS was used to culture all three trophoblast cell lines in an incubator at 37°C and in an atmosphere of 5% CO2. The medium was changed when the confluency reached 50%. The cells were subjected to incubation for a set of time at 37°C, and the humidified atmosphere of the incubator contained 93% N2, 5% CO2, and 2% O2 (Invivo2 Hypoxia Workstation, Ruskinn Technology, Leeds, West Yorkshire, United Kingdom). For each experiment, the cells were subjected to culture in triplicate.
Flow cytometry (with antibodies obtained from eBioscience, San Diego, CA, United States, including CD34, CD105, CD73, CD90, CD44, CD45, IG1, and HLA-DR) was used to examine the expression of cell markers in AD-MSCs (passage 3); these markers included positive markers (CD44, CD73, CD90, and CD105) and negative markers (CD34, CD45, CD146, IG1, and HLA-DR).
Moreover, AD-MSCs from normal placentas or placentas with severe PE could differentiate into osteoblasts as well as adipocytes; therefore, we assessed their differentiation capability. AD-MSCs cultured in 6-well plates were grown to approximately 70–80% confluency. Then, the AD-MSCs were cultured in differentiation medium (osteogenic or adipogenic) (Gibco, Carlsbad, CA, United States) for 3 weeks. Alizarin red S was used to stain the AD-MSCs to verify osteoblast differentiation; for adipocyte differentiation, oil red O was selected.
Exosomes were obtained from epidural AD-MSC supernatants by differential centrifugation. The medium was discarded when AD-MSCs reached 70% confluency. Then, the cells were cultured in serum-free DMEM/F12 for another 24 h. The supernatants were collected and then cleared by sequential centrifugation at 15,000 ×
In approximately 10 min, almost 50 μl of prepared exosomes were adsorbed and placed onto formvar carbon-coated 300-mesh copper grids. Then, the adsorbed exosomes were dried at room temperature for 30 min and negatively dyed with 3% phosphotungstic acid. Later, by using a transmission electron microscope (Olympus Software Imaging Solutions) at 120.0 kV, the exosomes were examined. Moreover, a digital camera was used to capture images of the exosomes.
We purchased anti-microtubule-associated protein LC3, P62, CD63, and BECN1 (beclin1, an autophagosome initiator) antibodies from R&D Systems (Minneapolis, MI, United States). Cell Signaling Technology (Danvers, MA, United States) provided the following antibodies: anti-EZH2 (#5246), anti-mTOR (#2983), anti-p-mTOR (#5536), anti-S6K1 (#2708), anti-p-S6K1 (#9204), anti-TSG101 (#28405), and anti-Ki67 (#9449). GSK126 (EZH2inhibitor) (10 μM), PQR620 (50 nM) and bafilomycin A1 (Baf A1, an autophagosome-lysosome fusion inhibitor) (100 nM) were purchased from MedChem Express (Monmouth Junction, NJ, United States). EZH2 plasmids were purchased from Shanghai Genechem Co., Ltd.
AD-MSC-derived exosomes (10 μg/ml) was used to treat JEG-3, and HTR-8 cells for 24 h. Then, we isolated the total RNA from the trophoblasts with TRIzol Reagent (Takara, Japan). After that, a reverse transcription kit (Invitrogen) was used to synthesize complementary DNA. Master Mix (Thermo Fisher Scientific) and Gene-specific TaqMan probes (Applied Biosystems) were used to carry out quantitative real-time PCR (RT-PCR) according to the manufacturer’s instructions. The expression of each target gene was normalized to GAPDH expression. We used TaqMan probes for EZH2 (Hs00544830_m1), mTOR (Hs00234508_m1), S6K1 (Hs00356367_m1), and GAPDH (Hs02786624_g1), and conducted three separate reactions for each marker.
Short interfering RNA (siRNA) oligonucleotide duplexes targeting EZH2 used in this study were synthesized and purified by RiboBio (Ribobio Co., Guangzhou, China). The sequences are as follows: siEZH2 #1: 5′-GCUGGAAUCAAAGGAUACA-3′; siEZH2 #2: 5′-GCGTTTCTTGTATCGGGAAAT-3′. A nonsense siRNA with no homology to the known genes in human cells was used as negative control: 5′−UUC UCC GAA CGUGUC ACG UTT−3′. Transfections of siRNA in trophoblasts were performed by using Lipofectamine 2,000 (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s instructions, and the knockdown efficiency was verified 48 h after transfection. All the siRNAs were used at a final concentration of 100 nM.
To make EZH2 constructs and its mutants, the human EZH2 gene was amplified by PCR and cloned into the SgfI/MluI sites of the pCMV6-Entry vector (Life Technologies).
We added trophoblasts to ninety-six-well plates (density: 5,000 cells per well), cultured these cells, and measured trophoblast proliferation daily via CCK-8 assays (Thermo Fisher Scientific, Waltham, MA, United States). We added CCK-8 reagent to each well, and cultured the trophoblasts for another 1.5 h. Then, colorimetric assays were performed by measuring the absorbance [optical density (OD) value] of each well in a microplate reader (wavelength: 450 nm). The growth curves were ascertained in three separate experiments.
According to the manufacturer’s instructions (Guangzhou RiboBio, Guangzhou, China), 5-Ethynyl-2’-deoxyuridine (EdU) assays were conducted by using a Cell-Light EdU
Trophoblasts were lysed on ice for 12 min with RIPA buffer (Sigma, St. Louis, MO, United States). After centrifugation at 12,000 ×
Subsequently, primary rabbit monoclonal antibodies against human LC3, BECN1, P62, EZH2, mTOR, p-mTOR, S6K1, and p-S6K1 (1:1,000 dilution) or β-actin (same dilution; Proteintech, Chicago, IL, United States) were incubated with the blocked membranes. Then, secondary antibodies were incubated with the membranes (1:1,000; CST, Danvers, MA, United States). The protein-antibody complexes were detected and quantified by using a chemiluminescence detection system (Bio-Rad, Hercules, CA, United States).
All placentas were collected after operation, treated within 30 min and closely examined for any visible abnormalities. After thorough rinsing with PBS 3 times to remove the maternal blood, the placental villous tissues were chopped into 8-mm3 pieces (2 mm × 2 mm × 2 mm). DMEM/F12 (4 ml per well) with 1% penicillin/streptomycin and amphotericin B (Gibco, Carlsbad, CA, United States) was used to culture the placental explants in six-well dishes (Corning) in a hypoxic incubator for 48 h at 37°C in an atmosphere of 2% oxygen. After AD-MSC-derived exosomes treatment for 24 h, PBS was used to rinse the explants; after that, they were frozen in liquid nitrogen.
Paraformaldehyde (4%) was used to fix the human term placental explants for 60 min. We embedded the tissues in paraffin, sliced them into 4-μm sections, and deparaffinized them. Then, the slides were boiled in 6.0 pH sodium citrate buffer (10 mM) for 7 min at 120°C for antigen retrieval. Hydrogen peroxide was used to block endogenous peroxidase for 10 min. We subsequently washed the slides three times for 5 min each with TBS (containing 0.05% Tween 20) (TBS/T; Merck; Darmstadt, Germany); later, these slides were incubated with monoclonal anti-EZH2 antibodies (1:200) for 12 h at 4°C. Diluted biotin-labeled secondary antibodies were incubated with these sections for 20 min at 37°C. We visualized the target proteins via fresh DAB solution and used hematoxylin as a tissue counterstain. Using an optical microscope (Olympus FV500, Tokyo, Japan), the expression of the target proteins was assessed by two observers independently. The staining area and intensity in five different random regions (200× magnification) were analyzed with Image-Pro Plus 5.1 to assess the protein expression levels.
Trophoblasts were cultured with or without purified AD-MSC-derived exosomes for 24 h (10 μg/ml). Then, all the cells were gathered and separated, and later for 60 min, they were fixed with 4% paraformaldehyde. The fixed cells were cut into 4-μm sections and embedded in paraffin. Later, they were washed in PBS for three times and blocked with 10% goat serum for 1 h. After that in 0.2% Triton X-100 the sections were washed twice. Next, they were incubated with primary antibodies (anti-LC3-II and anti-tubulin were purchased from Abcam, Cambridge, MA, United States), secondary antibodies (Invitrogen) and DAPI (Guangzhou RiboBio, Guangzhou, China). Using a fluorescence microscope, images were captured.
One-way ANOVA or two-tailed Student’s
First, we isolated AD-MSCs from healthy placentas, and identified their multidirectional differentiation ability and surface marker expression (
AD-MSC-derived exosomes promote the autophagy and proliferation of trophoblasts in hypoxia condition.
To better understand the changes in trophoblasts that occurred after AD-MSC-derived exosome treatment, we compared the EZH2 levels in the two trophoblast cell lines cultured for 48 h with or without AD-MSC-derived exosomes under hypoxic conditions. The mRNA levels of EZH2 significantly decreased in trophoblasts treated with AD-MSC-derived exosomes (
After confirming that AD-MSC-derived exosomes activated autophagy and inhibited the EZH2 expression in trophoblasts, we investigated whether EZH2 could regulate the trophoblast autophagy. Trophoblasts were treated with the specific EZH2 inhibitor GSK126 and autophagy inhibitor Baf A1. The results confirmed the inhibiting effective concentration of GSK126 on the H3k27me3 levels in trophoblasts (
The inhibition of EZH2 induced the increase of trophoblast autophagy and inhibition of mTOR pathway under hypoxic conditions.
The knockdown of EZH2 induced the increase of trophoblast autophagy and mTOR pathway inhibition under hypoxic conditions.
First, we confirmed that the overexpression plasmids of EZH2 could elevate the EZH2 mRNA levels in trophoblasts (
EZH2 overexpression decreased trophoblast autophagy and proliferation through mTOR signaling pathway.
To assess whether EZH2 mediated trophoblasts autophagy inhibition through the mTOR signaling pathway, the trophoblasts were transfected with EZH2-overexpression plasmids and treated with PQR620, a highly potent and selective mTOR inhibitor. Then, the expression levels of EZH2, mTOR and autophagy associated proteins in trophoblasts were evaluated by Western blotting. The Western blotting results confirmed that EZH2 overexpression in trophoblasts activated mTOR signaling and inhibited the autophagy in trophoblasts, and mTOR inhibitor attenuated the EZH2-mediated autophagy inhibition (
EZH2 regulate trophoblast autophagy and proliferation through mTOR signaling pathway.
Next, the data from trophoblasts were compared with those from placental explant cultures, which are
AD-MSC-mediated EZH2 activation increased autophagy in placental explants under hypoxic conditions.
Taken together, these data suggest that AD-MSC-derived exosomes inhibit the EZH2 expression and mTOR signaling pathway, and increase autophagic activity in cultured placental explants.
The amnion is the innermost avascular layer of the embryonic membranes and is an important source of promising cells that have therapeutic value. It has been used to improve a variety of medical conditions such as ophthalmology, skin injuries, and premature ovarian insufficiency. Its therapeutic effects are attributed to its inflammation-counteracting and immunomodulatory properties. Although the exact mechanisms are not clear yet, factors secreted by AD-MSCs are suggested to be the great contributors. In spite of the fact that the pathological mechanism of PE is illusive, dysfunction and hypoxia in trophoblasts were thought to be associated with preeclamptic placentas (
Autophagy was once deemed to occur in cytoplasm; however, recently, more and more evidences suggest that nuclear machineries (transcription factors, histone modification, microRNAs, etc.) are also involved in autophagy regulation (
The mechanistic target of rapamycin (formerly mTOR, mammalian target of rapamycin) is a serine/threonine protein kinase that is evolutionarily conserved, playing a central role in regulating the cell growth, proliferation and survival, according to the condition of nutrition, signals of stress and growth factors. It is a crucial controller of fundamental biological processes including lipid and glucose metabolism, autophagy, apoptosis, etc. Many researches have identified mTOR as a vital autophagy regulator, and deregulations of the mTOR pathway have been found to be involved in various pathological failures. On a molecular basis, autophagy regulates several signaling pathways that determines the death or continued survival of cells; nevertheless, the relationship between autophagy, EZH2 and mTOR pathways in trophoblasts is still undecided. The fate of trophoblasts, which were involved in hypertensive diseases during pregnancy, was affected by the autophagy regulation by AD-MSC-derived exosomes. In this study, the EZH2 expression was found to be reduced in trophoblasts treated with AD-MSC-derived exosomes, accompanied by the inactivation of the mTOR signaling pathway. These findings confirmed the curative effects of AD-MSC secretions and provided a novel insight into potential PE therapies.
In summary, it is revealed in our study that AD-MSCs promote the trophoblast proliferation and autophagy under hypoxic conditions
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
The studies involving human participants were reviewed and approved by The Affiliated Hospital of Qingdao University. The patients/participants provided their written informed consent to participate in this study.
YC, YH, and YY conceived and designed the experiments. WC, YoL, YZ, JuZ, and HZ performed the experiments. WP, LX, JiZ, NZ, GG, and XW collected the samples. LL, JL, RH, CL, and KY analyzed the data. YC, HZ, and YaL wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank the Department of Obstetrics and Gynecology, The Affiliated Hospital of Qingdao University, Qingdao, China, for providing tissue samples.
The Supplementary Material for this article can be found online at:
Characterization of primary AD-MSCs derived from human placental tissues.
Effect of EZH2 siRNA, EZH2 inhibitor and overexpression plasmids on the EZH2 and mTOR pathways proteins expression of trophoblasts by western blot.
Levels of LC3-II in EZH2 siRNA, inhibitor and overexpression plasmids treated trophoblasts with or without ADSCs/Baf A1 by Immunofluorescence assays.