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. 2020 Jan 13;36(5):363–370. doi: 10.1002/kjm2.12178

Mesenchymal stem cells‐derived exosomes improve pregnancy outcome through inducing maternal tolerance to the allogeneic fetus in abortion‐prone mating mouse

Yan‐Jie Xiang 1,2, Yan‐Yan Hou 3, Hong‐Li Yan 2, Hui Liu 4, Yan‐Xin Ge 4, Na Chen 5, Jian‐Feng Xiang 6,✉, Cui‐Fang Hao 7,✉
PMCID: PMC11896352  PMID: 31943723

Abstract

Recurrent pregnancy loss (RPL) is three or more times of consecutive spontaneous loss of pregnancy. The underlying cause is complicated and the etiology of over 50% of RPL patients is unclear. In the present study, bone marrow mesenchymal stem cells were isolated from CBA/J female mice and exosomes were isolated from cell culture medium by ultracentrifugation. CBA/J female mice were paired with male DBA/2 to generate abortion prone mouse model, and CBA/J females paired with male BALB/c mice were used as control. Exosomes were injected through uterine horns into pregnant CBA/J mice on day 4.5 of gestation in abortion‐prone matting. On day 13.5 of pregnancy, abortion rates were calculated and the level of transforming growth factor‐β (TGF‐β), interleukin 10 (IL‐10), interferon g (IFN‐γ), and tumor necrosis factor a (TNF‐α) in CD4+ T cells and macrophages in deciduas were evaluated by flow cytometry. Exosomes injection improved the pregnancy outcomes in abortion prone mice. The IL‐4 and IL‐10 levels on CD4+ T cells were upregulated in the maternal–fetal interface; meanwhile, the TNF‐α and IFN‐γ levels on CD4+ T cells were reduced. The IL‐10 level was increased and IL‐12 was reduced on the monocytes that separated from deciduas. miR‐101 level was increased in the CD4+ T cells in the deciduas. In conclusion, the treatment of ESCs‐derived exosomes modulates T cells' function and macrophages activities in the maternal–fetal interface that resulted in a decreased embryo resorption rate, and provides a therapeutic potential to treat RPL.

Keywords: exosome, mesenchymal stem cell, recurrent pregnancy loss

1. INTRODUCTION

Recurrent pregnancy loss (RPL) is defined as three or more consecutive spontaneous loss of pregnancy that affects approximately 1%‐5% of pregnant women.1 Until now, the etiology of about 50% patients with RPL is unknown. Pregnancy is a well‐choreographed physiological process and the pregnancy outcomes is affected by many factors involving immune tolerance, angiogenesis, hormonal balance, genetic, and epigenetic factors.2, 3, 4 Emerging evidences indicate that immunological disorders are among the main causes of RPL.

The abortion‐prone model of CBA/J × DBA/2 mating was used for recurrent abortion research since 1994, with an abortion rate between 20% and 40%.5, 6 Evidences from the CBA/J × DBA/2 model indicated that successful pregnancy was dependent on limitation of maternal immunity, especially suppressing the expression of T helper (TH)1‐type immunity and enhancing TH2‐type immune responses.7, 8 Meanwhile, the spontaneous abortion in this model was identified to relate to systemic maternal immune inflammation, increased complement deposition, and overactivation of NK cells and T cells in the maternal–fetal interface.9, 10

Mesenchymal stem cells (MSCs) are multipotent stromal cells that have been found as an important immunoregulator through cell‐cell interaction and cytokine secretion.11, 12, 13 It is reported that MSCs can promote pro‐inflammatory M1 macrophages transformed to anti‐inflammatory M2 macrophages.14 Additionally, MSCs can secrete cytokines and growth factors such as IL‐10, TGFβ1, and prostaglandin E2 (PGE2) that repress the inflammation.15 Recently, MSCs have been shown to inhibit the immune responses during pregnancy and the injection of MSCs via tail vein or uterine horns decreases abortion rate in mouse models.16, 17

Exosomes are membrane‐coated small vesicles secreted from host cells that can deliver functional molecules, including proteins and nucleotides, into recipient cells. The exosomes derived from MSCs have been found can attenuate the immune response in type 1 Diabetes and uveoretinitis mouse models but their function during pregnancy is still unknown.18 However, the effects of MSCs derived exosomes on spontaneous abortion are still unknown. In this study, exosomes were isolated from the MSCs culture medium and injected into CBA/J female mice through uterine horn to examine the effects of MSCs‐derived exosomes on embryo resorption. Reduced activities of T cells and macrophages were found in the maternal–fetal interface that contribute to a decreased embryo resorption rate.

2. MATERIALS AND METHODS

2.1. Mice

All experiments procedures on animals were carried out in accordance with the Guiding principles for research involving animals and human beings. Ethical approval was obtained from the Shandong University Animal Ethics Committee before the start of the study. Female CBA/J, male DBA/2, and male BALB/c mice (6‐8 weeks) were purchased from Beijing HFK Bioscience Co. Ltd., China and kept under pathogen‐free conditions in an animal house and fed normal mouse chow, and given tap water ad libitum.

2.2. BMSC isolation and culture

Bone marrow mesenchymal stem cells (BMSCs) were isolated form CBA/J female mice as previously described.19 Briefly, mice were sacrificed and then the femur and tibia were collected. Both ends of the femur and tibia were removed to expose the bone marrow cavity, which was flushed out by 0.9% normal saline. After centrifugation at 1500 g/min for 10 minutes, the cell pellet was collected, and the cells were resuspended in C57BL/6 mouse BMSC medium (Cyagen Biosciences, Santa Clara, California) containing 10% fetal bovine serum (FBS). Cells were cultured at 37 in the presence of 5% CO2, and the medium was refreshed after 72 hours and every 3 days thereafter were purified with CD11b (Microglia) MicroBeads (Miltenyi, Auburn, California), and cultured for further use.

2.3. Exosome isolation

Exosomes were isolated from cell culture medium by ultracentrifugation which was previous described.20 Briefly, cell culture medium was harvested and centrifuged at 300g at 4°C for 10 minutes to remove cells. The supernatant was collected, filtered through 0.22 μM filters and then centrifuged at 100 000g at 4°C for 2 hours. The exosome containing pellets were resuspended by PBS and then centrifuged at 100 000g at 4°C for 2 hours again, to obtain the exosome pallets.

2.4. Exosome injection and pregnancy outcome

CBA/J female mice were paired with male DBA/2 or BALB/c mice and checked for the vaginal plugs formation every morning. The day of vaginal plug detection was identified as day 0.5 of the pregnancy. Female CBA/J × male DBA/2 mating was used as the abortion‐prone mouse model (n = 5).21 The BALB/c mated CBA/J females that was used as normal pregnant group (n = 5). Under flutothane inhalational anesthesia, laparotomy was done and the pelvic region was exposed. All groups were injected PBS or exosomes through uterine horns at day 4.5 of pregnancy (implantation window). On day 13.5 of pregnancy, the females were sacrifice mice by CO2 asphyxiation, their uteri were removed and the implantation sites and resorption sites were documented. The abortion rate was calculated as the ratio of resorption sites and total implantation sites.

2.5. Transmission electron microscopy

The exosomes were fixed with 2% paraformaldehyde was prepared to fix the EVs at 4°C for 10 min, and subsequently transferred to carbon‐coated 200 mesh copper grid. Adsorption was allowed to occur for 5 minutes at 4°C. The grids were washed twice with PBS, followed by eight washes with deionized water. The grids were counterstained with uranyl acetate (2%) for 1 minute at 4°C, and subsequently air‐dried. Negative staining of exosomes was observed on the basis of their shape, structure, and size using a Philips CM12 transmission electron microscope at ×80 000 magnification (Philips Research China, Ltd., Shanghai, China).

2.6. RNA extraction and quantitative reverse polymerase chain reaction (qRT‐PCR)

Trizol reagent (Invitrogen) was used to extract RNA from all the samples according to the manufacturer's instructions. Briefly, samples were resuspended with 100 μL PBS and then mixed with 1 mL Trizol. After mixed with 200 μL chloroform and centrifugation, the supernatants were transferred into a new tube and the RNAs were precipitated by isopropanol. The RNA pellets were washed by 70% ethanol and then resolved. The RNA concentration and purity were determined using a model ND‐2000 spectrophotometer (Nanodrop Technologies, Wilmington, Delaware). Only samples with absorbance ratios 260 nm/280 nm of ~2.0 and 260 nm/230 nm of 1.9‐2.2 were considered for inclusion in the study.

The levels of miRNAs were detected by TaqMan miRNA RT‐Real Time PCR. Single‐stranded cDNA was synthesized using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, California) and then subjected to qPCR using miRNA‐specific TaqMan MGB probes (Applied Biosystems). U6 snRNA was used for normalization in cells. Each sample in each group was measured in triplicate and the experiment was repeated at least three times.

2.7. Spleen cells isolation

The spleen was aseptically removed and mechanically teased out of the stroma in PBS. The cell suspensions were isolated through 100 μm‐pore size nylon mesh and then treated with NH4Cl/Tris buffer to remove red blood cells. Subsequently, the cells were washed three times and prepared for in vitro culture in complete medium of RPMI 1640 containing 10% FBS, 1 mM l‐glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin.

2.8. Isolation of decidual immune cells

The uteruses and placentas of mice were removed and minced on ice, and the tissues were digested and isolated with collagen I and Dispase (Invitrogen). The cell suspensions were collected and cultured in RPMI 1640 complete medium with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin in 5% CO2 at 37°C. After primary culture for 2 hours at 37°C, the nonadherent decidual immune cells were collected while adherent decidual stromal cells were discarded.

2.9. Flow cytometry analysis

Expression of cell surface molecules and intracellular cytokines were evaluated using flow cytometry. MSCs were incubated with biotin‐labeled anti‐mouse CD29 (BioLegend) or CD105 (BioLegend) or CD45 (BioLegend) or SCA‐1 (BioLegend) or TER119 (BioLegend) or CD11B (BioLegend) antibody for 1 hour at 4°C and then incubated with PE labeled anti‐Biotin antibody.

The immune cells were incubated with biotin labeled anti‐mouse IL‐4 or IL‐10 or TNF‐α or IFN‐γ or CD86 or CD206 or IL‐12 antibody for 1 hour at 4°C and then incubated with PE labeled anti‐Biotin antibody. Cell fluorescence was determined by flow cytometry.

2.10. Immunoblotting

All protein samples were denatured by boiling in sodium dodecyl sulfate/β‐mercaptoethanol loading buffer and then were separated using 10% PAGE GEL. The proteins in the gels were blotted onto a polyvinylidene fluoride membrane (Amersham Pharmacia Biotech, St. Albans, Herts, UK) by electrophoretic transfer and then incubated with one of the primary antibodies overnight at 4°C after blocking by 5% nonfat milk. The membranes were incubated with horseradish peroxidase conjugated secondary antibody for another 2 hours at room temperature and then the signals were detected by using ECL kit (Pierce, Appleton, Wisconsin).

Anti‐CD63 antibody (ab59479, Abcam, California); anti‐Tsg101 antibody (ab30871, Abcam, California); anti‐calnexin antibody (#2433, Cell Signaling Technology, Inc., Danvers, Massachusetts).

2.11. Statistical analysis

Statistical analysis was performed using the SPSS software version 19.0 (IBM Corp., Armonk, New York). All results were analyzed by Students' t test and two‐tailed P < .05 was considered to indicate a statistically significant difference.

3. RESULTS

3.1. MSCs and exosomes isolation

To investigate whether exosomes from MSCs have beneficial effects on the pregnancy loss in vivo, MSCs were isolated from BALB/c mice and then confirmed by flow cytometry after surface marker staining (Figure 1A). The exosomes were isolated from the cell culture medium by ultracentrifugation and then subjected to transmission electron microscopy detection and immunoblotting. Figure 1B shows the typical extracted exosome which further confirmed by examine exosome marker: CD63 and Tsg101 through immunoblotting (Figure 1C).

Figure 1.

Figure 1

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Immunophenotype of bone marrow mesenchymal stem cells (BMSCs) and exosomes isolation. A, BMSCs were isolated form CBA/J female mice and phenotypically assessed for the expression of stem cell specific markers. Histograms show a representative of flow cytometric analysis of surface markers of MSCs. The red presented as the isotype controls. B, The exosomes were isolated from the MSCs culture medium by ultracentrifugation and then subjected to transmission electron microscopy. C, Immunoblotting was used to confirm the isolation of exosomes by detecting exosome markers: CD63 and Tsg101

3.2. Exosomes injection improved the pregnancy outcomes in abortion prone mice

Exosomes from MSCs were injected into the CBA/J female mice through uterine horn at day 4.5 of pregnancy (implantation window) and then the implantation rates were evaluated on day 13.5 of gestation. It is reported that the abortion rate in DBA/2 mated CBA/J female mice ranged from 20% to 40%.5 In the present study, the spontaneous embryo resorption rate is 24.6% (Figure 2). Meanwhile, the resorption rate was significantly reduced in the exosome injection group (11.5%) when compared with DBA/2 × CBA/J control group, but not significantly changed when compared with normal pregnancy model (CBA/J × BALB/c group; 8.34%; Figure 2). Moreover, there was no significant difference in the number of total embryos per uterus among these groups, indicating that exosomes from MSCs improved the pregnancy outcome via suppressing the fetal loss. Subsequently, we detected the CD4+, CD8+, and CD19+ cells by flow cytometry. We did not found significant differences of CD4+ and CD19+ cells among these three groups (Figure 2C,E). Increased CD8+ cells were found in CBA/J × DBA/2 group, which was reduced by exosome injection (Figure 2D).

Figure 2.

Figure 2

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The injection of mesenchymal stem cells (MSCs)‐derived exosomes reduced the embryos resorption rate. MSCs derived exosomes were injected into CBA/J female mice through uterine horn at day 4.5 of pregnancy (implantation window). The embryo resorption rates were calculated on day of 13.5 of gestation (n = 5 in each group). The expression of IL‐4, IL‐10, IFN‐γ, and TNF‐α mRNA levels in the decidual tissues was measured using quantitative real‐time PCR. Results were analyzed by Student's t test and P < .05 was considered significant. Error bars depict the SEM. *P < .05, **P < .01

3.3. Exosomes injection has no system effect on T cells and macrophages

Th2 bias is important at the maternal–fetal interface during a successful pregnancy, and Th1 bias is considered to be one of the reason for an abortion‐prone pregnancy.22 Based on mentioned results, we hypothesized that exosomes from MSCs may promote a Th2 shift from Th1, providing a compatible environment for a successful pregnancy at the maternal–fetal interface.

T cells and macrophages were isolated from the spleen to analyze whether exosomes play the regulatory role systemically or locally. As shown in Figure 3, there was no significant difference of IL‐4, IL‐10, TNF‐α, and IFN‐γ levels in CD4+ T cells between exosome treated or nontreated groups based on CBA/J × DBA/2 model (P > .05). Meanwhile, no significant difference of CD86, CD206, IL‐10, and IL‐12 levels in macrophages was observed in these groups (P > .05). These results indicated that exosomes injection has no effect in systemically modifying phenotypes of CD4+ cells and macrophages.

Figure 3.

Figure 3

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The injection of MSCs derived exosomes through uterine horns has no effect on CD4+ T cells and macrophage of the spleen. The splenic lymphocytes were stimulated with PMA(50 ng/mL), ionomycin(1 μg/mL), and BFA(10 μg/mL). For intracellular staining, cells were fixed and permeabilized, incubated with antibodies, and then analyzed by flow cytometry. The data are presented as mean ± SEM. Results were analyzed by Student's t test and P < .05 was considered as significant. NS means no significance

3.4. Exosomes injection skews toward a Th2 bias at the maternal–fetal interface

To further understand whether MSCs exosomes have effects in the maternal–fetal interface, we isolated the CD4+ T cells and macrophages in the deciduas. After detection by flow cytometry, we found exosomes injection resulted in a downregulation of TNF‐α and IFN‐γ and upregulation of IL‐4 and IL‐10 in the CD4+ T cells in the maternal–fetal interface, and their levels were similar to those in the normal mating group (CBA/J × DBA/2) (Figure 4). In the macrophages, exosomes upregulated IL‐10 and downregulated IL‐12 but had no effect on the expression of CD86 and CD206(Figure 4). These results indicated that exosomes injection through uterine horns suppresses the Th1 immune response and facilitates Th2 immune response, which generated the Th2 dominance in the immune microenvironment and could be beneficial to the pregnancy.

Figure 4.

Figure 4

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The injection of MSCs derived exosomes through uterine horns induced CD4+ T cells and macrophages of the decidua toward immunosuppressive phenotype. The decidual immune cells were isolated, cultured, and then analyzed by flow cytometry after incubation with different antibodies. The data are presented as mean ± SEM. Results were analyzed by Student's t test and P < .05 was considered as significant. NS means no significance. *P < .05, **P < .01 compared with CBA/J × DBA/2 group. #P < .05, ##P < .01 compared with normal pregnancy mating CBA/J × BALB/c group

3.5. Exosomes delivered miR‐101 into T cells

It is reported that exosomes can deliver miRNAs from host cells to recipient cells. So, we hypothesized that the exosomes from MSCs may regulate T cells differentiation through delivering miRNAs. So, we detected six miRNAs in the CD4+ T cells separated from the deciduas. These six miRNAs were reported related to recurrent pregnancy loss.23 As shown in Figure 5, the miR‐101 level was increased 7.2‐fold in the exosome injection group, indicating exosome delivered miR‐101 may contribute to the Th2 bias.

Figure 5.

Figure 5

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Exosomes from mesenchymal stem cells (MSCs) delivered miR‐101 into T cells. CD4+ T cells separated from the deciduas and subjected to RNA extraction. The level of six candidate miRNAs was quantified by quantitative reverse polymerase chain reaction (qRT‐PCR) and the results were analyzed by one‐way ANOVA. P < .05 was considered as significant. *P < .05, **P < .01

4. DISCUSSION

In this study, CBA/J × DBA/2 mating model was used to examine the effects of MSCs derived exosomes on spontaneous abortion. The abortion rate of control group is 24.6%, which is a suitable model for this study.

It is already confirmed that MSCs can secrete inflammation repressive cytokines and growth factors, and play immune responses inhibitor role when injected into abortion prone mice, which decrease the abortion rate.15, 16, 17 Although many reports suggest that MSCs application is safe and has beneficial effects during the treatment of autoimmune and chronic inflammatory diseases, the differentiation potential and ability to promote tumor growth still provide concerns for MSCs clinical use.24, 25, 26 MSC‐derived exosomes has been found can deliver functional proteins and microRNAs that modulate both physiological and pathological processes such as organism development, epigenetic regulation, immunoregulation; however, their roles during pregnancy is still unknown.27, 28 In the present study, we estimated the effects of ESC‐derived exosomes during pregnancy via uterine horns injection. The macrophages at the maternal–fetal interface promoted the IL‐10 release and decreased the secretion of IL‐12 after exosomes treatment, which manifested an anti‐inflammatory M2 type. This result was consistent with the former study that treated abortion prone mice by MSCs treatment.16

It has been shown that a Th2 bias at the maternal–fetal interface is necessary for a successful pregnancy, whereas the Th1 bias is found contributes to the abortion‐prone pregnancy.29 In this study, exosomes injection through uterine horns downregulated the production of IFN‐γ and TNF‐α, and upregulated IL‐10 and IL‐4, which indicated that ESCs‐derived exosomes changed the local immune microenvironment and decreased the embryo resorption rate.

miR‐101 was found downregulated in the maternal plasma of RPL patients.30 It is reported that MiR‐101 is highly represented in human naïve CD4+ T cells and its transfection into the EL4 murine T cell line downregulates inducible co‐stimulatory molecule (ICOS) and related to autoimmunity.31 In the present study, we observed reduced miR‐101 level in the CD4+ T cells from abortion prone mice indicating important roles of miR‐101 in the Th1 bias. However, miR‐101 is a multiple function molecule and may have tens to hundreds target genes. The mechanisms of miR‐101 in the T cells differentiation during pregnancy needs to be further investigated.

In conclusion, the treatment of ESCs‐derived exosomes modulates T cells' function and macrophages activities in the maternal–fetal interface, which resulted in a decreased embryo resorption rate, and provides a therapeutic potential to treat recurrent pregnancy loss.

CONFLICT OF INTEREST

All authors declare no conflict of interest.

Xiang Y‐J, Hou Y‐Y, Yan H‐L, et al. Mesenchymal stem cells‐derived exosomes improve pregnancy outcome through inducing maternal tolerance to the allogeneic fetus in abortion‐prone mating mouse. Kaohsiung J Med Sci. 2020;36:36:363–370. 10.1002/kjm2.12178

Yan‐Jie Xiang and Yan‐Yan Hou contributed equally to this study.

Contributor Information

Jian‐Feng Xiang, Email: xiangjf2009@163.com.

Cui‐Fang Hao, Email: cuifang_hao765@aliyun.com.

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