Extracellular vesicles secreted by human uterine stromal cells regulate decidualization, angiogenesis, and trophoblast differentiation
Edited by Thomas Spencer, University of Missouri, Columbia, MO; received January 6, 2022; accepted July 16, 2022
Significance
A major challenge in reproductive sciences today is to decipher the molecular basis of cell–cell communication during early pregnancy. The present study reveals that extracellular vesicles (EVs) generated by human endometrial stromal cells carry a variety of molecular cargo that augments decidualization, stimulates angiogenesis, and modulates trophoblast differentiation. We also show that the pleiotropic functions of EVs support communication between various cell types within the uterus that is vital in promoting decidualization, angiogenesis, and trophoblast development.
Abstract
In humans, the uterus undergoes a dramatic transformation to form an endometrial stroma-derived secretory tissue, termed decidua, during early pregnancy. The decidua secretes various factors that act in an autocrine/paracrine manner to promote stromal differentiation, facilitate maternal angiogenesis, and influence trophoblast differentiation and development, which are critical for the formation of a functional placenta. Here, we investigated the mechanisms by which decidual cells communicate with each other and with other cell types within the uterine milieu. We discovered that primary human endometrial stromal cells (HESCs) secrete extracellular vesicles (EVs) during decidualization and that this process is controlled by a conserved HIF2α-RAB27B pathway. Mass spectrometry revealed that the decidual EVs harbor a variety of protein cargo, including cell signaling molecules, growth modulators, metabolic regulators, and factors controlling endothelial cell expansion and remodeling. We tested the hypothesis that EVs secreted by the decidual cells mediate functional communications between various cell types within the uterus. We demonstrated that the internalization of EVs, specifically those carrying the glucose transporter 1 (GLUT1), promotes glucose uptake in recipient HESCs, supporting and advancing the decidualization program. Additionally, delivery of HESC-derived EVs into human endothelial cells stimulated their proliferation and led to enhanced vascular network formation. Strikingly, stromal EVs also promoted the differentiation of trophoblast stem cells into the extravillous trophoblast lineage. Collectively, these findings provide a deeper understanding of the pleiotropic roles played by EVs secreted by the decidual cells to ensure coordination of endometrial differentiation and angiogenesis with trophoblast function during the progressive phases of decidualization and placentation.
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During early pregnancy in humans, the endometrium undergoes a critical functional transformation orchestrated by the ovarian steroid hormones that allows embryo implantation. This transition must be synchronized with embryonic development to ensure maximal reproductive success (1–4). To enable this synchronization, an intricate maternal–fetal dialogue has evolved permitting the developing embryo and the endometrium to be in constant communication with each other (3, 4). As pregnancy progresses, the endometrial stromal cells undergo differentiation to form the decidua, a stroma-derived secretory tissue (2–4). It produces and secretes paracrine factors that control diverse endometrial functions, including the formation of an extensive vascular network that supports embryo development (5, 6). Proper differentiation of the trophoblast cells, critical for forming a functional placenta, is also influenced by yet unknown maternal factors secreted by the decidual cells. Any disruption in these processes may hinder placenta development, resulting in various diseases of pregnancy such as recurrent miscarriage, preeclampsia, and intrauterine growth restriction (7–10). A major challenge in reproductive sciences today is to understand the complex mechanisms by which signaling factors produced by the decidual cells are communicated to other cells within the uterine environment to influence dynamic adaptive processes, such as angiogenesis and maternal–fetal crosstalk, during early pregnancy.
A growing body of evidence indicates that extracellular vesicles (EVs) are essential mediators of cell–cell communication that underlie tissue functions at various physiological states (11–15). EVs are membrane-enclosed vesicles, which are secreted from the plasma membrane into the extracellular space. They include exosomes, which are typically 30 to 200 nm in diameter, and microvesicles, which range from 200 nm to 1 µm in diameter. Several different types of cargo, including proteins, lipids, and nucleic acids, can be found within these vesicles. As EVs are shed by one cell and taken up by another, these cargoes are transferred to the recipient cells and alter their functions (11–16). As described in this paper, we recently observed that human endometrial stromal cells secrete abundant EVs into culture media during in vitro decidualization. This observation prompted us to consider the hypothesis that EVs secreted by the decidual cells mediate cell signaling and communication between various cell types within the pregnant uterus. One predicts that a defect in decidual EV secretion is likely to impair critical processes during early pregnancy. Indeed, recent reports implicated EVs in pregnancy maintenance. Their secretion from first-trimester placenta led to the postulation that they mediate maternal–fetal interactions during pregnancy (17, 18).
However, the mechanisms that control the secretion of EVs and their precise roles in human decidual tissue have not been elucidated. In this study, we demonstrate that a pathway involving HIF2α, a hypoxia-inducible factor, and RAB27B, a member of the Rab family of GTPases, operates in primary human endometrial stromal cells (HESCs) to promote the production and secretion of EVs during decidualization. We also characterized the protein cargoes carried by these EVs and analyzed their functional roles in changing recipient cell function. Our study reveals that EVs secreted by HESC play a critical role in controlling the efficient progress of stromal differentiation in an autocrine manner and mediate stromal–endothelial and stromal–trophoblast communications, contributing to the fundamental understanding of the paracrine signaling mechanisms that control progressive phases of decidualization, angiogenesis, and placentation.
RESULTS
A Conserved HIF2α-RAB27B Pathway Regulates EV Secretion by HESCs.
Our previous studies in mice revealed that the transcription factor hypoxia‐inducible factor 2 alpha (HIF2α), which is induced in response to the physiologically hypoxic conditions that exist within the uterus during early pregnancy, is an essential regulator of implantation (19). Mice conditionally lacking Hif2α in the endometrium exhibited down-regulation of Rab27b, which displays GTPase activity and is a regulator of protein trafficking. RAB27B is reported to regulate the secretion of EVs in certain human cell lines in vitro (20). We, therefore, set out to examine whether the HIF2α-RAB27B pathway operates in primary HESCs and whether it promotes EV secretion by these cells.
To study decidualization, we typically employ a well-characterized in vitro cell culture system in which HESCs undergo decidualization in response to a differentiation mixture (DC) containing 1 µM progesterone (P), 10 nM 17-β-estradiol (E), and 0.5 mM 8-bromo-adenosine-3′,5′-cyclic monophosphate (8-Br-cAMP). As shown in Fig. 1A, HIF2α transcripts are induced in HESCs during in vitro decidualization when cells are cultured under hypoxic conditions. When HIF2α expression is down-regulated by administering small interfering RNAs (siRNAs) specifically targeted to these transcripts (Fig. 1B), we observed a corresponding reduction in RAB27B messenger RNA (mRNA) levels (Fig. 1C) and RAB27B protein expression as seen by Western blotting and immunocytochemistry (Fig. 1 D–F). Taken together, these results indicated that a HIF2α-RAB27B pathway operates in HESC during decidualization.
Fig. 1.
We next examined whether HESCs indeed secrete EVs during decidualization. The cells were allowed to undergo decidualization for 72 h, conditioned medium was collected at the end of this period, and EVs were isolated using the miRCURY (Qiagen) kit. The EV pellet was then resuspended and analyzed by transmission electron microscopy (TEM) imaging, which allowed us to visualize the vesicles. The EVs displayed a relatively homogeneous morphology (Fig. 2A, Left panel). As shown in Fig. 2B, the size range of the vesicles spanned from 30 to 100 nm in diameter, which is within the typical range for exosomes. We further validated the EVs by using Western blot analysis to detect well-known EV biomarkers, including tetraspanins CD63 and CD81 as well as cytosolic protein Tsg101 (Fig. 2C).
Fig. 2.
To determine whether decidualization impacts on EV secretion, we compared the extent of EV secretion by HESCs into conditioned media with or without the addition of DC. Using quantitation by microfluidic resistive pulse sensing (MRPS) analysis, we observed that the production of EVs by decidualizing HESCs was dramatically elevated (∼10-fold) relative to those secreted by undifferentiated HESCs (Fig. 3A). We also examined the effects of the individual components of DC on EV production and secretion. As shown in SI Appendix, Fig. S1, treatment with E alone had only minimal effects on EV secretion. While treatment with P or 8-Br-cAMP alone was able to promote EV production and secretion, we observed maximal effects only when all three components of DC were present together.
Fig. 3.
We also observed that hypoxia, compared to normoxic conditions, facilitates EV secretion by decidualizing HESCs as shown in Fig. 3B. We next examined whether EV secretion by HESCs during decidualization under hypoxic conditions is controlled by HIF2α-RAB27B. siRNA-mediated down-regulation of either HIF2α or RAB27B transcripts led to a marked reduction in EV secretion by HESCs as evidenced by TEM analysis (Fig. 3C). This was further confirmed by quantitation of EVs secreted into conditioned media by decidualizing HESCs under hypoxic conditions (Fig. 3D). Clearly, the loss of HIF2α or RAB27B transcripts in HESCs severely impaired EV secretion by these cells. Collectively, these results indicated that a conserved HIF2α-RAB27B pathway operates in HESCs during decidualization to regulate vesicular trafficking associated with EV secretion.
Mass Spectrometry Revealed that EVs Secreted by HESCs Carry a Variety of Cargo Proteins with a Wide Range of Functions.
The purity of EVs is crucial for accurate identification and characterization of their cargo proteins. We combined two well-characterized methods to purify the EVs. We first isolated EVs from cell culture media using the miRCURY Exosome kit (Qiagen). The harvested EVs were then washed with phosphate-buffered saline (PBS) and subjected to ultracentrifugation to remove any contaminating proteins. To further characterize the EVs, we employed an affinity-based method. We used antibodies against specific tetraspanin proteins, namely, CD63 and CD81, which are localized on the EV membrane, and performed flow cytometry to characterize the EVs bound to the antibody-coated magnetic beads (SI Appendix, Fig. S2). The EV–bead complexes were detected with APC-conjugated anti-CD81 antibody and PE-conjugated anti-CD63 antibody. We found that beads incubated with EVs showed an increased intensity of CD63 and CD81 when compared to naive beads, which confirmed that our purification method indeed isolates bona fide EVs containing specific tetraspanin protein biomarkers on their surfaces.
We next isolated EVs from the conditioned media of HESCs undergoing in vitro decidualization for 24, 48, 72, and 96 h. The purified EVs were subjected to mass spectrometry to identify their protein components/cargoes (SI Appendix, Tables S1–S4). Prominent among these are structural proteins, including various tetraspanins (e.g., CD9, CD63, and CD81) and several annexins, which are known biomarkers of EVs. We also identified several RAB GTPases, Syndecan-2, and Syntenin-1, which are involved in EV biogenesis. Our results also revealed the presence of potentially functional cargo proteins, which can potentially modulate a variety of uterine cell functions during decidualization. These include several factors controlling endothelial cell expansion and remodeling (e.g., ANGPT1, ANGPTL2, ADAM10, ADAM9, ADAMTS1, GJA1, matrix metallopeptidase 2 [MMP2], TIMP1, TIMP2, RAC1, and several integrins), metabolic regulators (e.g., GLUT1, PKM, and SLC1A5) controlling stromal differentiation, and growth modulators (e.g., IGF2 and insulin-like growth factor binding proteins (IGFBP) IGFBP1, IGFBP3, IGFBP5, IGFBP7) that are potential regulators of trophoblast differentiation (Table 1).
Table 1.
| Gene name | Protein description |
|---|---|
| Factors potentially involved in EV biogenesis and trafficking | |
| ANXA1 | Annexin A1 |
| ANXA2 | Annexin A2 |
| ANXA5 | Annexin A5 |
| ANXA6 | Annexin A6 |
| ANXA11 | Annexin A11 |
| CD44 | CD44 antigen |
| CD81 | Tetraspanin |
| CD9 | Tetraspanin |
| CD63 | Tetraspanin |
| FLOT2 | Flotillin-2 |
| PDCD6IP | Programmed cell death 6-interacting protein |
| RAB10 | Ras-related protein Rab-10 |
| RAB1A | Ras-related protein Rab-1A |
| RAB5C | Ras-related protein Rab-5C |
| RAB6A | Ras-related protein Rab-6A |
| RAB7A | Ras-related protein Rab-7a |
| RAP1B | Ras-related protein Rap-1b |
| RHOA | Transforming protein RhoA |
| SDC2 | Syndecan-2 |
| SDCBP | Syntenin-1 |
| Potential regulators of angiogenesis | |
| ANGPT1 | Angiopoietin-1 |
| ANGPTL2 | Angiopoietin-related protein 2 |
| ADAM10 | Disintegrin and metalloproteinase domain-containing protein 10 |
| ADAM9 | Disintegrin and metalloproteinase domain-containing protein 9 |
| ADAMTS1 | A disintegrin and metalloproteinase with thrombospondin motifs 1 |
| GJA1 | Gap junction alpha-1 protein |
| ITGA1 | Integrin alpha-1 |
| ITGA2 | Integrin alpha-2 |
| ITGA6 | Integrin alpha-6 |
| ITGAV | Integrin alpha v |
| ITGB1 | Integrin beta-1 |
| LOXL2 | Lysyl oxidase homolog 2 |
| MMP2 | 72 kDa type IV collagenase |
| TIMP1 | TIMP metallopeptidase inhibitor 1 |
| TIMP2 | TIMP metallopeptidase inhibitor 2 |
| RAC1 | Ras-related C3 botulinum toxin substrate 1 |
| Potential regulators of decidualization | |
| DCN | Decorin |
| GLUT1 | Glucose transporter type 1 |
| MFGE8 | Lactadherin |
| PKM | Pyruvate kinase |
| Potential regulators of trophoblast differentiation | |
| IGFBP1 | Insulin-like growth factor-binding protein 1 |
| IGFBP3 | Insulin-like growth factor-binding protein 3 |
| IGFBP5 | Insulin-like growth factor-binding protein 5 |
| IGFBP7 | Insulin-like growth factor-binding protein 7 |
| SLC1A5 | Amino acid transporter |
| IGF2 | Insulin-like growth factor II |
| TGFBI | Transforming growth factor-beta-induced protein ig-h3 |
| TGFB1 | Transforming growth factor beta 1 |
*The complete list is provided in SI Appendix, Tables S1–S4.
Our results indicated that the complexity of the EV cargo increased as decidualization progressed from 24 to 96 h (SI Appendix, Tables S1–S4). While 236 different protein cargoes were identified at 24 h, we found 377 proteins at 96 h. Protein abundance was assessed based on label-free quantitative liquid chromatography–mass spectrometry (LC-MS) and compared between EVs collected at different time points to determine relative changes in selected cargoes with the progression of the decidualization program (SI Appendix, Fig. S3). The resulting heatmap indicated that many proteins, such as GLUT1, MMP2, TIMP1, and TIMP2, became increasingly abundant with the progression of decidualization. This pattern was further confirmed for GLUT1 and MMP2 by Western blot analysis using antibodies against these proteins (SI Appendix, Fig. S4). Other protein cargoes displayed a different pattern of distribution. For example, the abundance of certain proteins, such as ADAM10, ITGB1, IGF2, and SDC2, peaked at 72 h and then declined at 96 h.
EVs Internalized by Endometrial Stromal Cells Enhance Their Differentiation Potency.
We next performed experiments to ascertain that the EVs secreted by the differentiating stromal cells can be internalized by recipient cells. For this purpose, the lipid membranes of isolated EVs were labeled using a PKH26 fluorescent dye. The labeled EVs were added to HESC culture, and their uptake was monitored over a 24-h period by confocal microscopy. We could directly visualize that EVs were internalized by HESCs (Fig. 4A, Right). In contrast, no PKH26 fluorescence was detected in cells incubated with the dye alone without labeled EVs (Fig. 4A, Left). Using z-stack image analysis, we further confirmed the localization of the EVs in the cytoplasm of recipient stromal cells, confirming that they are indeed internalized by these cells (Fig. 4B).
Fig. 4.
Based on our mass spectrometry findings, we hypothesized that certain EV protein cargo would affect specific cellular functions in recipient uterine cells upon incorporation into these cells. To test our hypothesis, we first investigated the effects of the addition of EVs on the decidualization capacity of recipient HESCs. In this experiment, we isolated EVs from the conditioned media of differentiating stromal cells and then added increasing concentrations of EVs to undifferentiated HESCs in the presence of the DC and monitored decidualization at 0 and 48 h. We then monitored the gene expression profiles of various differentiation biomarkers at these time points. As expected, we noted increased mRNA levels of various decidualization markers, including IGFBP1, PRL, and HAND2, in differentiating HESCs at 48 h (Fig. 5, columns N/A at 0 and 48 h). However, in response to the addition of EVs, the expression of these genes was further enhanced at 48 h of in vitro decidualization (Fig. 5). These results indicated that EVs promote the decidualization potency of recipient stromal cells, confirming our hypothesis that their cargo has distinct functional effects on these cells.
Fig. 5.
GLUT1 Is a Functional Protein Cargo Carried by Endometrial Stromal EVs.
As indicated in Table 1, we identified glucose transporter 1 (GLUT1), a membrane protein involved in glucose transport, by the two independent methods for determining EV contents. Because EVs are taken up by recipient cells, we postulated that their GLUT1 cargo would be incorporated into recipient cells to facilitate the entry of glucose in these cells. Glucose will then be metabolized to provide the energy required to support decidualization. As reported previously by Neff et al. (21), GLUT1 mRNA is expressed as HESCs undergo decidualization in vitro. Interestingly, addition of EVs did not alter the level of GLUT1 mRNA (Fig. 6A) but increased the GLUT1 protein level in these cells as indicated by Western blot analysis (Fig. 6B). The observation that the GLUT1 protein level increased without a concomitant mRNA increase likely reflects the incorporation of EVs with GLUT1 cargo proteins directly into the HESC membranes.
Fig. 6.
To ascertain the role of GLUT1 carried by EVs, we employed an RNA interference approach, using siRNA to knockdown endogenous GLUT1 mRNA levels in HESCs, followed by DC stimulation. At 48 h posttransfection with siRNA specifically targeting GLUT1 mRNA, we observed more than an 80% reduction in GLUT1 mRNA in HESCs compared with that in response to nontargeting siRNA (Fig. 6C). We next isolated EVs from stromal cells with or without siRNA treatment and added them to GLUT1-depleted HESC cultures subjected to in vitro decidualization. As shown in Fig. 6D, the addition of increasing amounts of exogenous EVs isolated from HESCs treated with control siRNA led to significant enhancement of the expression of differentiation markers, IGFBP1 and PRL. Remarkably, when we added EVs depleted of GLUT1 cargo via treatment of HESC with GLUT1 siRNA, we did not observe any significant change in IGFBP1 and PRL transcript levels (Fig. 6D). These results indicated that the GLUT1 cargo in the EVs taken up by the HESCs contributes significantly to the decidualization process, presumably by promoting glucose uptake and its subsequent metabolism to provide energy.
We next assessed whether the internalization of stromal EVs can indeed influence glucose uptake by differentiating HESCs. We treated HESCs with or without EVs following the initiation of differentiation with DC and monitored the time course of glucose uptake by these cells. Fluorescence-conjugated 2-NbDG, a glucose analog, was used to track the incorporation of glucose. As shown in Fig. 6E, upon addition of exogenous EVs, we observed a progressive enhancement in 2-NbDG uptake by the recipient HESCs starting at ∼6 h in the decidualization program. We observed a remarkable ∼50% increase in EV-induced glucose uptake compared to the control as the decidualization process advanced to 24 h, and then this uptake increased further as the differentiation reached a peak at 48 h. These results showed that the GLUT1 cargo carried by the EVs can functionally mediate glucose transport and the resulting boost in energy metabolism positively impacts on the progression of the decidualization process.
Stromal EVs Promote In Vitro Angiogenesis.
We next examined whether HESC-derived EVs can influence endothelial cell functions. For this purpose, we first tested whether the stromal EVs are internalized by endothelial cells. PKH26-labeled EVs were added to cultures of human umbilical vein endothelial cells (HUVECs), and after 24 h of incubation, the cells were washed thoroughly and examined by confocal microscopy. As shown in Fig. 7A, EVs were taken up by the HUVECs. In contrast, no PKH26 fluorescence was detected in cells without added EVs (Fig. 7A).
Fig. 7.
We next investigated whether EV treatment affects HUVEC functions, such as their proliferation and angiogenic factor production. We therefore assessed the expression of Ki67, a positive proliferative marker expressed in cell nuclei, using immunocytochemistry. We noticed that the proliferation of HUVECs treated with EVs increased significantly compared to untreated cells (Fig. 7 B and C). We also noted a significant increase in the cytoplasmic expression of angiopoietin-2 protein, a well-known angiogenic marker, in these cells, indicating that the uptake of stromal EVs could augment the production of angiogenic factors by the endothelial cells, potentially promoting angiogenesis (Fig. 7 B and C).
Finally, we assessed in vitro angiogenesis by the tube formation assay of endothelial cells. HUVECs were cultured for 48 h in a growth medium supplemented with or without stromal EVs. We were able to visualize the capillary-like structures formed by HUVECs via Calcein-AM, which delineates the proliferating cells that make up new vascular structures (22). The HUVECs exposed to HESC-derived EVs exhibited significantly enhanced tube formation, after only 6 to 12 h, as assessed by the quantitation of tube length and loop numbers, compared to those grown in regular medium (Fig. 7 D and E). Collectively, these results indicated a clear functional effect of HESC-derived EVs on endothelial cell function during angiogenesis.
Impact of Stromal EVs on Trophoblast Differentiation.
To address whether EVs produced by HESCs could influence trophoblast function, we utilized an in vitro model system using human trophoblast stem cell (TSC) lines derived by Drs. Hiroaki Okae and Takahiro Arima. These cells can be maintained in a self-renewing stem state or can be efficiently differentiated to extravillous trophoblast (EVT) lineage (23, 24). As shown in Fig. 8, we have successfully induced the differentiation of these human TSCs into EVT cells. During this differentiation, the cells acquire distinct morphological characteristics (Fig. 8A) and progressively express HLA-G, MMP2, and ITGAV, known markers of EVT differentiation over a period of 8 d (Fig. 8B). Additionally, we confirmed that the EVTs are characterized by up-regulation of transcripts corresponding to CCR1, ERVW1, IGF2, HLAG, GCM1, and MMP2 and down-regulation of EPCAM (SI Appendix, Fig. S5). Remarkably, we found that the addition of EVs isolated from decidualizing HESCs to these TSC lines markedly enhanced their differentiation to EVT (Fig. 9 A–D). This was indicated by an increased expression of MMP2, HLA-G, and ITGAV, which was observed on both days 4 and 8 of the differentiation program (Fig. 9 A–D). These results indicated that EVs secreted by decidualizing endometrial stromal cells control trophoblast differentiation, thereby regulating the paracrine functionality of trophoblast cells.
Fig. 8.
Fig. 9.
DISCUSSION
For the past several years, studies in our laboratory as well as by others have focused on an important biological problem, namely, deciphering the molecular basis of cell–cell communication during embryo implantation and establishment of pregnancy. Clearly, intercellular communication between various uterine cell types plays a central role in controlling the physiology of early pregnancy. Earlier studies indicated that this can be mediated through direct cell-to-cell contact via gap junctions or transfer of secreted molecules between cells within the uterine milieu. We had previously shown that gap junction communication between adjacent stromal cells via connexin 43 plays an important role in endometrial angiogenesis and maintenance of pregnancy (25). We had also reported that fibroblast growth factors, secreted by endometrial stromal cells, act via a paracrine mechanism on epithelial cells to regulate uterine receptivity during implantation (26). In the present study, we have examined the emerging role of EVs in cell–cell communication within the uterine tissue during decidualization and identified several candidate factors that participate in this process. Our study reveals that EVs generated by the stromal cells carry critical molecular cargo that augments decidualization, stimulates angiogenesis, and modulates trophoblast differentiation.
It was previously reported that the secretion of EVs from plasma memebranes of HeLa cells into the extracellular space is controlled by certain members of the Rab GTPase family, Rab27a and Rab27b (20, 27). Our recent studies in mice showed that the expression of the Rab27b gene is regulated by Hif2α during implantation in mice (19). In the present study, we demonstrate that the induction of HIF2α in human endometrial stromal cells under hypoxic conditions during decidualization in vitro controls the expression of RAB27B, which regulates the trafficking and secretion of EVs. These results support the concept that the HIF2α-RAB27B pathway dictates critical and conserved signaling mechanisms that mediate adaptive functions in the human endometrium to influence the secretory activity of this tissue during decidualization and pregnancy establishment.
To investigate how HIF2α-RAB27B-directed EV secretion by the decidual stromal cells may influence uterine function, we embarked on the identification of their protein cargo, employing mass spectrometry. Analyzing EVs secreted at different days (24 to 96 h) during decidualization, we identified a range of EV cargo proteins that are potential regulators of critical events during decidualization, such as cell metabolism, cell proliferation and differentiation, and cell remodeling (Table 1 and SI Appendix, Tables S1–S4).
Most interestingly, our analyses identified GLUT1 as a prominent EV protein cargo. In successful pregnancy, glucose is the primary energy source for metabolic activities of both embryonic and maternal tissues. GLUT1, the main transporter responsible for glucose uptake and consumption, plays a critical role in decidualization, which demands increased energy metabolism (28). Previous studies in our laboratory showed that GLUT1 expression is progressively induced in HESCs during decidualization (21). Reduced expression of GLUT1 contributed to a loss of glucose uptake in HESCs and led to impaired differentiation. Consistent with these findings, it was reported that insufficient glucose metabolism during gestation results in placental defects and fetal growth restriction, leading to pregnancy disorders, such as intrauterine growth restriction (IUGR) (7, 8, 10). The discovery of GLUT1 in HESC-derived EVs opens up the possibility that the direct transfer of this glucose transporter between uterine cells plays a critical role in modulating glucose metabolism during progressive phases of decidualization. It has not escaped our attention that therapeutic administration of these transporters might be similarly achieved.
An important aspect of our study is the demonstration that stroma-derived EVs can be internalized by endometrial stromal, endothelial, and trophoblast cells. We noted that the GLUT1 protein levels of the recipient stromal cells increased following the uptake of EVs, indicating successful delivery of the protein cargo. These observations set the stage to study the downstream functional effects of the transfer of protein cargo to the recipient cells. We observed that the addition of EVs to HESC cultures significantly enhanced decidualization in vitro as evidenced by the elevated expression of multiple differentiation biomarkers, including IGFBP1, HAND2, and PRL (Fig. 5).
To further assess the specific impact of the GLUT1 cargo on the differentiation program of the recipient stromal cells, we employed a siRNA-mediated loss-of-function strategy. This led to a drastic depletion of endogenous GLUT1 in siRNA-treated HESCs, resulting in the production of EVs that lack GLUT1. Interestingly, when these EVs were added to differentiating HESCs, they failed to stimulate the differentiation program, establishing that EV-mediated intercellular delivery of GLUT1 contributes to the differentiation potency of HESCs. What is the biological rationale for the transfer of EVs with GLUT1 cargo from one stromal cell to another? We propose the following concept. As the decidualization program progresses spatiotemporally through the endometrium, it is conceivable that stromal cells that are in more advanced phases of the differentiation process may positively influence the energy metabolism in the next layer of stromal cells. This can be achieved via the direct transfer of the GLUT1 transporter from a cell that expresses ample GLUT1 to an adjacent layer of less differentiated stromal cells with a lower level of the transporter, thereby promoting the progressive ability of the recipient cells to take up glucose and advance their differentiation status. This concept finds strong support from the results shown in Fig. 6E, indicating that the addition of EVs to decidualizing stromal cells significantly accelerates the rate of their glucose uptake, providing the necessary energy that drives the progression of the decidualization process.
During human pregnancy, uterine angiogenesis is an essential physiological process since the formation of blood vessels is required for transporting nutrients across the maternal–fetal interface. Our previous study, using a mouse model, revealed that uterine neovascularization is supported via the induction of various stromal factors, such as VEGFs, Angiopoietin-2, Hif2α, MMPs, and Cx43 (25, 29, 30). Improper regulation of this process can lead to defective placental development and severe embryonic growth restriction (25, 30). Our present study provides evidence that HESC-derived EVs carry a variety of angiogenesis-regulating factors that may affect the proliferation and differentiation of vascular endothelial cells. As shown in Table 1, these EVs carry MMPs and their inhibitors TIMPs, which are known to be involved in the degradation of the extracellular matrix that facilitates remodeling at the stromal–endothelial interface (31, 32). The EV cargo also includes various other modulators of angiogenesis such as Angiopoietin-1, Angiopoietin-like 2, Lysyl oxidase-like 2 (LOXL2), ADAMTS-1, and ADAM10 (33–36). Furthermore, decorin, another stromal EV cargo, is reported to promote tube formation in endothelial cells, contributing to angiogenesis (37).
Consistent with these potential functions of EV cargo in angiogenesis, we demonstrated that EV-mediated signaling promotes the proliferation of endothelial cells and their differentiation to acquire the ability to produce critical angiogenic factors, such as Angiopoietin 2, a key autocrine regulator of vascular stability (Fig. 7). We further observed that the HESC-derived EVs, which are taken up by HUVECs, can promote the formation of capillary-like network structures by these cells. Overall, these in vitro studies confirmed that the HESC-derived EVs can mediate paracrine communication between stromal and endothelial cells to facilitate the induction of uterine angiogenesis.
A clear understanding of the molecular pathways that ensure the coordination of the endometrial differentiation and angiogenesis with embryonic growth during decidualization still eludes us. A two-way dialogue among endometrial stromal and trophoblast cells is considered essential for the establishment of pregnancy (38). Although several trophoblast-derived factors have been reported to guide trophoblast differentiation and invasion (38, 39), there is a severe lack of experimental models addressing the mechanisms by which maternal factors control trophoblast differentiation and invasion. Here, we utilized a human trophoblast differentiation system to show that the uptake of EVs secreted by the maternal tissue can influence trophoblast function. Our results strongly suggest that these EVs transport critical maternal factors to the trophoblast cells as the TSCs undergo differentiation into distinct trophoblast cell lineages. Specifically, we found that the addition of EVs isolated from decidualizing HESCs to TSCs markedly enhanced their differentiation to EVT. Interestingly, our mass spectrometry analysis revealed that HESC-derived EVs carry several IGFBPs, namely, IGFBP1, IGFBP3, IGFBP5, and IGFBP7 (Table 1). There is ample evidence that autocrine secretion of growth factors, specifically IGF1 and IGF-2, drives trophoblast proliferation and differentiation to invasive phenotype, whereas maternal decidua secretes IGFBPs that resist this invasive process (40–42). We are tempted to postulate that EV-derived IGFBPs act to neutralize IGF bioavailability and regulate IGF signaling, potentially contributing to the differentiation of TSCs to different EVT lineages. Further experiments are needed to test this hypothesis.
Although our knowledge is limited regarding the individual contributions of the EV cargo that facilitate angiogenesis and trophoblast differentiation, our future studies will continue to analyze the roles of multiple candidate protein cargoes in modulating the angiogenic pathways and trophoblast function. Our current findings, therefore, provide a solid basis that will help further analysis and understanding of the pleiotropic functions of EVs in supporting communication between various cell types within the uterus that is vital in promoting decidualization, angiogenesis, and trophoblast development during early pregnancy.
MATERIALS AND METHODS
HESC Culture.
Our studies used deidentified primary HESCs collected according to the guidelines set forth for the protection of human subjects participating in clinical research and are approved by the institutional review board of Wake Forest School of Medicine. Samples were collected from the early proliferative stage of the menstrual cycle by Pipelle biopsy from fertile, regularly cycling women under anesthesia before laparoscopy as described previously (24). Donors displayed no signs of endometrial pathological conditions and provided written informed consent. The subjects ranged in age from 28 to 42 y and in parity from 1 to 2 (21).
HESCs were cultured in Dulbecco’s modified Eagle medium (DMEM)/F-12 medium (Gibco) supplemented with 5% fetal bovine serum (FBS) (Atlanta Biologicals), 50 μg/mL penicillin, and 50 μg/mL streptomycin (Invitrogen) (21). To induce in vitro differentiation, the cells were treated with a DC containing 0.5 mM 8-Br-cAMP (Sigma-Aldrich), 1 μM P (Sigma-Aldrich), and 10 nM E (Sigma-Aldrich) in DMEM/F-12 medium (Gibco) supplemented with 2% (vol/vol) charcoal dextran–stripped FBS (Atlanta Biologicals) (21). HUVECs were purchased from PromoCell and maintained in standard endothelial cell growth medium (PromoCell). All cells were cultivated in a humidified incubator at 37 °C with 5% CO2. In certain experiments, HESCs were cultured under hypoxic (3% O2) conditions. The cells were subcultured once they reached about 70% confluency.
TSC Culture.
The cytotrophoblast-derived human TSC lines CT27 and CT29 were obtained from Dr. Michael Soares’ laboratory, University of Kansas Medical Center, under an agreement with Dr. Hiroaki Okae of Tohoku University who developed these lines (23, 24). These cells were cultured in 100-mm tissue culture dishes coated with 5 μg/mL mouse collagen IV (Corning) for ≥1.5 h at 37 °C. Stem cells were seeded at 0.5 × 105 to 1 × 105 cells/mL and maintained a highly proliferative state when cultured in complete media. Complete medium of stem cells consists of DMEM/F-12, 0.2% FBS, 0.3% fatty acid–free bovine serum albumin (BSA) (Thermo Fisher), 1% ITS-X (Thermo Fisher), 0.1 mM 2-mercaptoethanol (Sigma), 50 ng/mL recombinant hEGF (Sigma), 1.5 mg/mL L-ascorbic acid (Sigma), 0.8 mM valproic acid (Sigma), 5 µM Y27632 (Reprocell), 2 µM CHIR99021 (Reprocell), 0.5 µM A83-01 (Reprocell), 1 µM SB431542 (Reprocell), and penicillin–streptomycin (1×). Complete culture medium was replaced every 48 h. When stem cells reached 70 to 90% confluency, they were detached by TrypLE (Thermo Fisher) and passaged to new collagen IV–coated plates.
TSCs were induced to differentiate into EVT cells as described previously (23). Chamber slides (ibidi) or 6-well plates were coated by 1 to 2 µg/mL collagen IV at 37 °C for at least 1.5 h. Complete EVT medium for in vitro differentiation consists of DMEM/F-12, 0.3% BSA, 1% ITS-X, 0.1 mM 2-mercaptoethanol, 2.5 µM Y27632, 7.5 µM A83-01, 4% KnockOut Serum Replacement (Thermo Fisher), 100 ng/mL hNRG-1(Cell Signaling Technology), and penicillin–streptomycin (1×). On day 1, stem cells were dissociated by TrypLE and seeded at 0.5 × 105 to 1 × 105/mL in complete EVT media supplemented by 2% Matrigel (Corning). On day 3, EVT media were replaced without NRG1. On day 6, EVT media were replaced again without NRG1 or KnockOut Serum Replacement. EVT differentiation can be further enhanced for two additional days. Matrigel is necessary for the induction of EVT cells, which was supplemented in EVT media at 0.25 to 0.5% on both day 3 and day 6. Cells were analyzed on day 8 of EVT cell differentiation.
Isolation of EVs.
To harvest EVs, HESCs were washed with PBS and grown in serum-free DMEM/F-12 with the DC for 24 to 96 h. Conditioned medium was collected and centrifuged at 3,000 g for 10 min to obtain a cell-free specimen. The supernatant was further centrifuged at 16,500 g for 20 min at 4 °C to remove cell debris and apoptotic bodies. The supernatant was either stored at −80 °C or directly used to extract EVs using the miRCURY Exosome Cell/Urine/CSF kit (Qiagen) as described in the manufacturer’s protocol. The harvested EVs were further purified by ultracentrifugation to clear contaminating proteins. The EV pellet obtained by using the miRCURY kit was washed with PBS and centrifuged at 120,000 g for 90 min at 4 °C.
Quantitation of EVs by MRPS, TEM, Flow Cytometry of Evs Bound to Antibody-Coated Beads, siRNA Transfections, Mass Spectrometry, RNA Isolation and Quantitative PCR, Western Blot Analysis, Labeling of EVs by PKH26, Immunocytochemistry, Glucose Uptake Assay, Endothelial Tube Formation Assay, Statistical Analysis. Full details can be found in SI Appendix, SI Materials and Methods.
Data, Materials, and Software Availability
All study data are included in the article and/or SI Appendix.
Acknowledgments
This work was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/NIH R01 HD090066 (to I.C.B. and M.K.B.). We thank Drs. Peter Yau and Justine Arrington from the Roy J. Carver Biotechnology Center for performing mass spectrometry analysis.
Supporting Information
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Cover image: Pictured is the western end of Dauphin Island, Alabama. Heida L. Diefenderfer et al. examined the progress made over 10 years in restoring the coastal ecosystem along the Gulf of Mexico following the 2010 Deepwater Horizon oil spill. The authors report that nearly half of the authorized restoration funds were spent or committed by the end of 2021, and a comprehensive assessment of the collective effects of coastal restoration is needed to inform future expenditures. Although the restoration effort is in dire need of assessment to synthesize and delineate the multiple effects of large-scale restoration, the Gulf Coast region currently lacks an entity to conduct such a synthesis, according to the authors. See the article by Diefenderfer et al., e2213639119. Image credit: Bethany A. Carl Kraft.
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Copyright © 2022 the Author(s). Published by PNAS. This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).
Data, Materials, and Software Availability
All study data are included in the article and/or SI Appendix.
Submission history
Received: January 6, 2022
Accepted: July 16, 2022
Published online: September 12, 2022
Published in issue: September 20, 2022
Keywords
Acknowledgments
This work was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/NIH R01 HD090066 (to I.C.B. and M.K.B.). We thank Drs. Peter Yau and Justine Arrington from the Roy J. Carver Biotechnology Center for performing mass spectrometry analysis.
Notes
This article is a PNAS Direct Submission.
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Competing Interests
The authors declare no competing interest.
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