Dysregulation of Decidual NK Cell Proliferation by Impaired Decidual Cells: A Potential Contributor to Excessive Trophoblast Invasion in Placenta Accreta Spectrum

doi:10.21203/rs.3.rs-4849945/v1

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Research Article

Dysregulation of Decidual NK Cell Proliferation by Impaired Decidual Cells: A Potential Contributor to Excessive Trophoblast Invasion in Placenta Accreta Spectrum

https://doi.org/10.21203/rs.3.rs-4849945/v1

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Background

Abnormal interactions among decidual cells, decidual natural killer (dNK) cells, and trophoblasts are implicated in contributing to the placenta accreta spectrum (PAS). However, the specific details of these interactions remain unclear.

Methods

Normal human placental maternal decidua-mesenchymal stem cells (MD-MSCs) and pathological MD-MSCs from PAS patients (PAs) were isolated and cultured in serum-free conditions. Decidualization was induced using hormonal cocktails: estradiol (E2)/ progesterone (P4) and chemical agents 8-br-cAMP/ medroxyprogesterone acetate (MPA) for both MD-MSCs and PAs. dNK-like cells were generated from peripheral natural killer (pNK) cells through MD-MSCs induction. Interactions among decidual cells, dNK cells, and trophoblasts were studied using a transwell co-culture system. Bulk RNA-seq analysis was performed to identify differential genes between MD-MSCs and PAs and explored their potential role in immune tolerance regulation of decidual NK cells and trophoblast invasion.

Results

This study aims to explore the correlation between defective decidualization of endometrial stromal cells and dysregulated dNK cell proliferation, leading to excessive trophoblast invasion and the development of PAS. Decidualization defects were confirmed in PAs, characterized by reduced morphological changes and altered expression levels of decidual biomarkers at both mRNA and protein levels, potentially associated with overexpression of estrogen receptor (ER). Furthermore, both PAs and normal MD-MSCs exhibited similar patterns in regulating trophoblast invasion, suggesting an indirect impact of impaired decidual cells on trophoblast behavior. Interestingly, decidualized MD-MSCs (De-MD-MSCs) showed the potential to induce conversion of pNK cells into dNK-like cells, which displayed reduced cytotoxicity on trophoblasts and enhanced KIR2DL4 expression, possibly through upregulated Csf3, Il1β, and Tgfb1. Additionally, dNK-like cells exhibited increased proliferation when co-cultured with PAs, regulated by Cxcl12, Il33, Tgfb1, Vegfa, and Vegfc, enhancing trophoblast invasion and spiral artery remodeling. Conditioned medium derived from PAs-induced dNK-like cells demonstrated a higher capacity to promote trophoblast invasion in a dose-dependent manner.

Conclusion

Abnormal proliferation of dNK cells induced by impaired decidual cells may contribute to the pathogenesis of PAS, providing valuable insights into its mechanisms and potential therapeutic interventions.

Decidualization

Placenta accreta spectrum

Trophoblast invasion

Decidual natural killer cell

Immune tolerance

In the process of embryos implanting in the uterus, the interaction between the maternal decidual cells, leukocyte subpopulations like decidual natural killer (dNK) cells, and blastocyst trophoblasts are critical for the healthy placental formation and successful pregnancy [1]. During blastocyst development, trophoblasts differentiate into cytotrophoblasts (CTBs) and syncytiotrophoblasts (SCTs), forming the primitive syncytium and lacunae [2]. CTs subsequently differentiate into STs and extravillous trophoblasts (EVTs) that build chorionic villi in the placenta [3]. Studies demonstrate that decidual cells and dNK cells regulate trophoblast invasion by secreting factors, maintaining a delicate balance in this process [4]. Decidual cells, part of the specialized endometrium during pregnancy, complete the decidual reaction post-implantation, supporting placentation through interactions with trophoblasts and the production of specific proteins and factors [5]. dNK cells exhibit distinct phenotypic and functional characteristics compared to peripheral blood NK (pNK) cells. Throughout pregnancy, dNK cells, with unique killer cell immunoglobulin-like receptors (KIR) expression and interactions with decidual cells, play a crucial role in immune tolerance at the maternal-fetal interface and placentation through interactions with EVTs [6]. The equilibrium among decidual cells, dNK cells, and trophoblast is vital in establishing an immune-regulated environment necessary for the placental formation and fetal development, contributing significantly to maternal-fetal immune tolerance [7–8].

Various placental disorders, including the high-risk placenta accreta spectrum (PAS), are caused by disruptions in the regulation between these cells [9]. PAS is an abnormal placental development condition that encompasses placenta accreta, increta, and percreta and is a major cause of severe maternal morbidity and mortality [10]. PAS is particularly daunting for pregnant women and obstetricians due to the difficulty in early detection and the complexity of treatment even after diagnosis. Recently, the prevalence rate of PAS has increased in recent decades, from 1 in 1,250 births in the 1980s to as many as 1 in 272 births today [11]. From the cytological perspective, studies suggest that defects in the decidualization of endometrial stromal cells (ESCs) [12–13], altered immune responses in maternal tissues [14–15], or a combination of both conditions [16–18] are proposed as underlying factors contributing to excessive invasion of EVT. This can result in the pathologic adherence of the placenta and subsequent severe bleeding during delivery [19–21]. However, due to the lack of appropriate animal models and even cellular models, studying the intricate mechanisms governing the aberrant placentation of PAS has presented challenges. Thus, the precise etiology of PAS remains incompletely understood, especially when investigating the early and mid-stages of human pregnancy, during which EVTs interact with decidual cells and dNK cells. Therefore, understanding the complex interactions among decidual cells, EVTs, and immune cells, particularly dNK cells within the uterus is essential for developing diagnostic and therapeutic approaches to address PAS and its potentially life-threatening complications.

In our preliminary work, we isolated a unique cell population derived from the choriodecidual interface, where the decidua (developed from the endometrium) and the chorion (developed from the trophoblast of the outer wall of the embryo) fuse together. These cells were identified from the precise decidua expressing two X chromosomes with the MSC markers and differentiation potentials, known as human placenta maternal decidua derived-mesenchymal stem cells (MD-MSCs), replacing the previously confusing origin and nomenclature of human placenta choriodecidual membrane-derived mesenchymal stem cells (pcMSCs) [22]. They have shown significant potential for cell therapy due to their anti-inflammatory, immunomodulatory, and tissue repair capabilities [23]. Beyond their roles in regenerative medicine, this study aims to explore the physiological functions of MD-MSCs in the endometrium to gain new insights into complex placental diseases. Consequently, we developed an MD-MSC-based decidualization and co-culture system. This advanced system facilitates the assessment of EVT invasion and dNK cell induction, thereby potentially overcoming the limitations of traditional in vitro methods.

In this study, we established an MD-MSC-based co-culture system and utilized pathological MD-MSCs from patients with PAS (PAs) to investigate the underlying mechanism of PAS. Our findings suggest that MD-MSCs can potentially induce pNK cells to convert into dNK-like cells with lower cytolytic ability and enhanced expression of KIR2DL4, one of the polymorphic KIRs that interacts with HLA-G on EVTs [24]. Moreover, we observed that dNK-like cells proliferate significantly when co-cultured with PAs and exhibit a greater capacity to promote trophoblast invasion compared to co-cultured with MD-MSCs. This discovery highlights the potential contribution of aberrant dNK cell numbers, in addition to decidualization defects, to PAS development. This study not only proposes a novel approach to studying the intricate interactions among decidual cells, trophoblasts, and dNK cells in vitro, but offering new insights into the mechanism of PAS.

MD-MSCs and PAs isolation and culture

MD-MSCs and PAs cells were obtained from women undergoing cesarean sections at Taipei Medical University Hospital and Dr. Jin-Chung Shih from National Taiwan University Hospital, respectively. All procedures were approved by the Institutional Review Board (IRB: 202308101RINC). For cell isolation, the decidual membrane was isolated, chopped, and digested with a mixture of protease (Sigma, 9001-92-7), collagenase B (Sigma, COLLB-RO), and DNase I (Bioshop, DRB003). After overnight digestion at 4°C, the filtrate was centrifuged, washed, and resuspended in MCDB201 medium (Merck, M230428) supplemented with 1% insulin-transferrin-selenium (ScienCell, 0803), 10 ng/mL epidermal growth factor (EGF, Peprotech, AF10015), and 1% penicillin/streptomycin. Cells were seeded onto collagen-type IV-coated Petri dishes, and non-adherent cells were removed after 24 hours. Upon reaching 90% confluency, cells were dissociated with trypsin (Gibco, 25-200-072), terminated with fetal bovine serum (FBS) (Gibco, 10-082-147), and seeded for further amplification.

In vitro decidualization

For in vitro decidualization using (a) hormone and (b) second messenger induction, MD-MSCs or PAs were initially seeded in a 10-cm Petri dish and allowed to attach for 48 hours. Subsequently, the cells were stimulated with the combination of (a) 10 nM estradiol (E2, Sigma-Aldrich, E2785) and 1 µM progesterone (P4, Sigma-Aldrich, P8783) or (b) 0.5 mM 8-Bromo-cAMP sodium salt (MedChemExpress, HY-12306) and 1 µM medroxyprogesterone 17-acetate (MPA, Sigma-Aldrich, M1629) for 5 days. The morphology and the expression of decidualization markers were assessed at 5 days following induction. Throughout the decidualization process, the morphological changes of the cells were monitored using a phase-contrast microscope on the fifth day after induction. Furthermore, total protein and RNA were extracted from both decidualized cultures and non-stimulated control samples at specified time points for further analysis.

Decidual cell morphology quantification

MD-MSCs were initially seeded in a sterilized 12-well plate and subjected to two different decidualization cocktails. Following a 5-day decidualization period, cells were washed with PBS (pH 7.4) and fixed with 4% paraformaldehyde solution (BioLegend, 420801) in PBS for 10 minutes at room temperature. After fixation, cells underwent three PBS washes and were stained with Wheat Germ Agglutinin (WGA) (Invitrogen, W11261) at a concentration of 5 µg/mL to target the cell membrane, enhance visibility of cell boundaries, and facilitate cell morphology quantification. For nuclear visualization, DAPI (BioLegend, 422801) was applied at a concentration of 1 µg/mL for 5 minutes. Stained cells were then observed using fluorescence microscopy to capture images, and ImageJ software was employed for quantification of cell morphology. Circularities and roundness of 15 cells from each image were measured separately to assess cell shape, ensuring reliable statistical analysis.

Real-time PCR (RT-PCR)

Total RNA was extracted from MD-MSCs and PAs using the NucleoSpin® RNA isolation kit (740955.50, Macherey-Nagel). The concentration of RNA was determined using the Nanodrop 1000 (Thermo) spectrophotometer. Subsequently, reverse transcription was performed using the SuperScript first-strand synthesis system (Invitrogen, 48190-011) according to the manufacturer's instructions. The resulting cDNA samples were utilized for PCR amplification using the qPCRBIO SyGreen Blue Mix Lo-ROX (PB20.15). Quantitative PCR (qPCR) assays were conducted in triplicate on a Biometra Professional Basic 96 gradient detection system. To normalize the expression levels of target genes, the reference gene (18S rRNA) was used. The specific primers employed for each gene can be found in Table S1. The average threshold cycle (Ct) was calculated from the Ct values obtained from three replicates of each sample. The normalized ΔCt value for each sample was computed as the mean Ct of the target gene minus the mean Ct of the reference gene. ΔΔCt was then calculated as the difference between the ΔCt values of the control and test samples. The fold change in gene expression for each sample relative to the control was determined using the 2^(-ΔΔCt) mathematical model for relative quantification in qPCR. The mean fold induction and standard error of the mean (SEM) were determined from a minimum of three or more independent experiments.

Enzyme-linked immunosorbent assay (ELISA)

To measure the concentration of secreted proteins, such as prolactin (PRL) and Insulin-like growth factor binding protein 1 (IGFBP1), the cell culture medium was collected and centrifuged at 2,000g for 10 minutes at 4°C to remove any cellular debris. The resulting supernatant was immediately supplemented with a protease inhibitor cocktail and stored at -80°C for subsequent analysis. For the determination of scavenger receptor class A type 5 (SCARA5) protein levels, the cells were first lysed using RIPA buffer supplemented with a 100-fold diluted protease inhibitor cocktail. The cell lysate was then collected and centrifuged at 16,500g for 20 minutes. The resulting supernatant was transferred to a new Eppendorf tube and stored at -80°C until further use. To quantify the PRL and IGFBP1 secretion levels, specific ELISA kits were employed, such as the Human Prolactin DuoSet ELISA (R&D #DY682) and Human IGFBP1 DuoSet ELISA (R&D #DY871), in conjunction with the DuoSet ELISA Ancillary Reagent kit 2 (R&D #DY008B), following the manufacturer's instructions. Similarly, the SCARA5 levels were determined using an appropriate ELISA kit (AVIVA SYSTEMS BIOLOGY, OKCD00526).

Immunohistochemistry (IHC) in tissue sections

The human decidual tissue sections from normal and PAS placenta were formalin-fixed, paraffin-embedded, cut, and mounted at the Laboratory Animal Center, College of Medicine, National Taiwan University. The dissected sections were placed under 60°C to soften the paraffin for the following steps. After 30 minutes, the sections were dewaxed with Xylene-clear citrus oil (National Diagnostics, HS-200), cleared in 100% ethanol, and rehydrated through gradients of ethanol in PBS (with gradients of 100%, 95%, 80%). Antigen retrieval was then conducted in 100°C citrate acid buffer for 20 minutes. Sections were sequentially incubated with 1% blocking BSA for 5 minutes and subsequently incubated with primary antibodies at 4°C overnight. After washing with PBS to remove unbound antibodies, biotinylated secondary antibodies were applied. Following that, HRP polymer was applied for 30 minutes at room temperature. After 30 minutes, diaminobenzidine (DAB) substrate (Chromogen: Substrate = 1:20) was added for precipitation for 1–3 minutes. Hematoxylin was then used to stain cell nuclei. Sections were mounted with Malinol (MUTO PURE CHEMICALS,2040-2). The information on the antibodies used is provided in Table S2. IHC staining reagents were used following the manufacturer's instructions (Novolink, RE7290-k).

Surface marker expression levels measured by flow cytometry

For the evaluation of ER, PR, and SCARA5 expression levels in MD-MSCs and PAs, live cells were trypsinized and fixed with a 4% paraformaldehyde solution (BioLegend, 420801) in PBS for 10 minutes at room temperature. Permeabilization was achieved using 0.1% Triton X-100 for 5 minutes at room temperature. Subsequently, fixed cells were incubated individually with ER, PR, and SCARA5 antibodies at manufacturer-specified concentrations for 15 minutes. For surface CD3, CD56, KIR2DL4, and HLA-G expression levels, the same protocols were followed without Triton X-100 permeabilization. Finally, cells were resuspended in FACS buffer (1X PBS supplemented with 1 mM EDTA, 25 mM HEPES, and 1% FBS) for subsequent analysis. Analytical flow cytometry was performed using an LSRFortessa, and FACS data were analyzed using FlowJo (version 10.8.01).

Trophoblast invasion assay

To perform the invasion assay, MD-MSCs and PAs were seeded and cultured in a 24-well plate. Subsequently, these cells underwent treatment with two distinct decidualization cocktails. Following a 5-day decidualization, trophoblast cell line 3A-sub-E (BCRC, 60302) was seeded onto transwell inserts with an 8 µm pore size. These inserts had been pre-coated with a Matrigel solution (Corning, #356231) diluted sixfold in serum-free MEM α (Minimum Essential Medium) (Gibco, 11900024). After a 3-day co-culture period, the transwell inserts were meticulously removed, and non-invaded cells present on the upper surface of the inserts were gently eliminated using a cotton swab. Subsequently, the invaded trophoblasts were lysed utilizing a CellTiter-Glo® 2.0 Cell Viability Assay (Promega #G9241) to facilitate the quantification of the relative trophoblast invasion rate.

Peripheral blood mononuclear cells (PBMCs) isolation and peripheral blood natural killer (pNK) cells purification

pNK cells were obtained from human peripheral blood following approved procedures by the IRB (202312090RINC). Mononuclear cells were isolated from human peripheral blood using the Mitenyl Biotec protocol for density gradient centrifugation. Peripheral blood was collected in a 10 mL vacutainer containing heparin and centrifuged at 550g for 10 minutes to separate plasma from blood cells. The plasma was inactivated at 56°C for 30 minutes, followed by centrifugation at 3000g for 10 minutes to remove precipitate. For PBMC isolation, blood cells were diluted with four times the volume of commercial 1X PBS (Gibco, 00187) and layered over Ficoll-Paque (Cytiva, 17144002) in a 50 mL conical tube, then centrifuged at 400g for 40 minutes. The PBMC layer was carefully transferred to a new tube, washed with 1X PBS, and centrifuged at 300g for 10 minutes. Platelets were removed by resuspending the cell pellet in 1X PBS and centrifuging at 200g for 15 minutes, repeating the step twice. The resulting cell pellet was resuspended, and a cell count was performed. A total of 10⁷ cells were seeded in a T75 flask and cultured in a DSNK medium (BioMab, 020-A010-001) supplemented with inactivated plasma for pNK cell purification and expansion. After a 14-day selection period, the purity of pNK cells was assessed by flow cytometry, by determining the expression of the pNK cell marker CD3-CD56+. Once purity reached 95%, pNK cells were considered suitable for subsequent experiments.

Cytotoxicity assay for pNK cell and dNK-like cells

The pNK cells and dNK-like cells induced by MD-MSCs were prepared according to the aforementioned protocols. In the cytotoxic assay, 3A-subE trophoblasts were initially seeded into a 96-well plate. Following a 6-hour attachment period, the pNK cells and dNK-like cells were seeded to co-culture with the attached trophoblasts. The ratios of effector cells (NK cells) to target cells (trophoblasts) were set at five different proportions: 1:1, 2:1, 4:1, 1:2, and 1:4. After co-culturing for 12 hours, the NK cells were removed with the cell culture medium, and the remaining attached trophoblasts were lysed using the CellTiter-Glo® 2.0 Cell Viability Assay and measured for luminescence. The cytolytic ability of NK cells was quantified using the formula:

dNK-like cell proliferation assay

MD-MSCs and PAs were seeded in 96-well plates at a density of 5x10³ cells/well and cultured for 48 hours. These cells were then treated with two different decidualization cocktails. Following a 5-day decidualization period, purified pNK cells were seeded at a density of 5x10³ cells/well into the 96-well plates, establishing co-cultures with MD-MSCs, PAs, and the decidual cells. During the co-culture period, the suspended NK cells were collected at specific time points: 1, 4, 7, and 10 days. To quantify the proliferation of dNK-like cells, the CellTiter-Glo® 2.0 Cell Viability Assay (Promega #G9241) was employed. This assay allows for the measurement of cell viability based on ATP levels, providing insights into cell proliferation and overall cellular health.

RNA-sequencing and data analysis

MD-MSCs and PAs were cultured in 10 cm Petri dishes. Following treatment with various decidualization cocktails, the total RNA of these cells was extracted using the NucleoSpin® RNA isolation kit (740955.50, Macherey-Nagel) and subsequently utilized for RNA-seq analysis. The RNA-seq procedure was carried out by BIOTOOLS (Taiwan). The genetic data obtained was analyzed using the Biotools RNAseq version 1.6.6 platform. This analysis yielded fold changes, Log2 (fold changes), and p-values for each gene, allowing for the identification of differentially expressed genes (DEGs) based on Log2 (fold changes) > 1 and p-values < 0.05. The values presented on the heatmap are expressed as z-scores, which have been normalized relative to the gene expression levels. Gene expression plots were generated using the website SRplot, while Venn diagrams were created using Venny2.0.

Statistical analysis

Statistical analysis was performed using GraphPad Prism version 9.1.1 (GraphPad Software, La Jolla, CA, USA). The data were presented as mean ± SEM (standard error of the mean). Statistical comparisons were conducted using one-way ANOVA with Dunnett's post hoc correction or two-way ANOVA followed by Tukey's or Bonferroni's post hoc tests. A significance level of P < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, n.s.: no significance).

MD-MSCs represent a distinct cell type with the capability for in vitro decidualization.

In this study, we have discovered a particular variety of MSCs, known as MD-MSCs. These cells were meticulously extracted from the decidual membrane of the human placenta, a process accomplished through serum-free culture techniques. In addition, they displayed a remarkable capacity for amplification in vitro, with the ability to undergo approximately 20 passages [22]. Given their inherent expression of estrogen (ER) and progesterone receptors (PR), we hypothesized that they could respond to hormonal stimuli and undergo decidualization (Fig. 1A).

To thoroughly assess the efficacy of the transition from MD-MSCs to decidual cells, we employed two well-established metrics: alterations in cell morphology and the upregulation of specific genes. Following a five-day induction period with hormonal (E2/P4) or chemical (cAMP/MPA) agents (Fig. 1A), MD-MSCs underwent significant morphological transformations, transitioning from a spindle-like to a rounded shape, as illustrated in Fig. 1B-a. To precisely quantify these observed changes in shape, we applied circularity and roundness parameters (Fig. 1B-b) [25]. Remarkably, upon exposure to the second messenger induction, three distinct batches of MD-MSCs exhibited a pronounced shift in morphology (Fig. S1A).

To further investigate the genetic changes associated with decidualization in MD-MSCs, we conducted an RNA-seq analysis to explore potential decidual cell markers. We compared our dataset with two previously published single-cell RNA-seq databases [26–27], aiming to identify upregulated genes that could serve as reliable biomarkers for decidual cells. This analysis resulted in the identification of 33 annotated genes (Fig. 1C), the expression patterns of which were then visualized in heatmap format, as depicted in Fig. 1D (Supplementary Data 1). Furthermore, 9 annotated genes are further investigated as potential decidual cell markers through comparison with the TISSUES database (BTO:0002770) (Supplementary Data 1). To validate the results obtained from RNA-seq, we conducted qPCR analysis on these 9 annotated genes (Fig. 1E). Interestingly, our findings highlighted three genes—prolactin (Prl), insulin-like growth factor binding protein 1 (Igfbp1), and scavenger receptor class A type 5 (Scara5)—as potential biomarkers for decidual cells. These potential decidual cell markers were selected based on a 10-fold change observed in both E2/P4 and cAMP/MPA treatments to avoid the induction of nonspecific upregulation through chemical stimulation. Previous studies have shown that MSCs from either bone marrow or adipose tissue could also undergo decidualization through cAMP/MPA treatment. However, it's important to note that the cAMP signaling pathway can bypass the ER and PR, directly driving downstream processes and promoting nonspecific signal transduction [28]. To address this concern, we also employed other types of MSCs to distinguish the decidualization potential of MD-MSCs. As anticipated, following E2/P4 treatment, the mRNA expression levels of Prl, Igfbp1, and Scara5 were dramatically upregulated in MD-MSCs, compared with bone marrow-derived MSCs (BMMSCs) and adipose tissue-derived MSCs (Fig. S1B). Furthermore, the protein expression levels of these three decidual cell markers measured by ELISA were consistent (Fig. 1F).

Decidualization defects in PAs have been observed in the MD-MSC-based decidualization system

Building upon previous research providing solid evidence for the decidualization potential of MD-MSCs, we aimed to delve deeper into the mechanisms underlying PAS, a condition associated with decidualization defects [29]. By utilizing PAs, we sought to unravel the detailed mechanisms of this disease through the decidualization system. We hypothesized that PAs might exhibit an inadequate degree of decidualization, rendering them unable to effectively resist trophoblast over-invasion [30]. First, as the representative images showed, we observed that PAs displayed fewer morphological changes compared to MD-MSCs (Fig. 2A-a). Quantitative analysis revealed that PAs retained their spindle shape rather than adopting a round shape, as indicated by their low roundness and circularity values (Fig. 2A-b). Additionally, we assessed the mRNA (Fig. 2B) and protein (Fig. 2C-D) expression levels of three annotated decidual cell marker genes in PAs. As the figures showed, the mRNA and protein expression levels of Prl and Igfbp1 were consistently reduced in two decidualized-PAs (De-PAs) when compared to De-MD-MSCs. The protein expression levels exhibited a similar trend in immunohistochemistry staining, with reduced levels in PAS compared with normal placenta. Conversely, the gene expression levels of Scara5 were higher in De-PAs but reduced in protein levels. The inconsistency in SCARA5 levels between mRNA and protein remains unknown due to the unclear function and involved signaling pathways. Nevertheless, these observations indicate a clear distinction in gene expression between PAs and MD-MSCs, suggesting an abnormal decidualization process in PAs. Furthermore, annotated genes upregulated during decidualization exhibit downregulation in De-PAs treated with E2/P4 (Fig. S2A). Therefore, we revisited the expression levels of ER and PR. Surprisingly, we discovered that ER was more expressed in PAs than in normal MD-MSCs, as evidenced by flow cytometry analysis (Fig. S2B). This finding was further corroborated by immunohistochemical (IHC) staining performed on PAS and normal placenta biopsy samples (Fig. S2C). In the severe pattern of PAS, the decidua is notably absent. Consequently, we selectively procured biopsies demonstrating moderate PAS, characterized by the presence of residual decidua, in IHC staining.

Decidualization defects may not constitute the primary cause of dysregulation in trophoblast invasion, thereby leading to PAS.

Considering the association between PAS and dysregulated decidual cells impacting trophoblast invasion, we established a co-culture system to evaluate the interaction between PAs and trophoblasts. The trophoblast invasion timeline is depicted in Fig. 3A. In this system, we made a surprising discovery, as illustrated in Fig. 3B. We found that MD-MSCs treated with cAMP/MPA inhibited trophoblast invasion, while normal and E2/P4-treated MD-MSCs enhanced trophoblast invasion. This implies that MD-MSCs and De-MD-MSCs can dynamically regulate trophoblast invasion, aligning with the concept proposed in clinical research that "decidual cells act as a natural stop signal to inhibit excessive trophoblast invasion." This phenomenon highlights the intricate interactions between decidual cells and trophoblasts and provides valuable insights into the regulation of trophoblast invasion during pregnancy.

Contrary to our expectations, both PAs and De-PAs demonstrated a similar pattern in regulating trophoblast invasion, as statistically analyzed across three batches of MD-MSCs (Fig. 3B). This finding challenges the assumption that impaired decidualization is one of the primary causes of PAS. Nevertheless, we investigated potential regulators and signaling pathways involved in trophoblast invasion regulation. The expression levels of 22 annotated genes associated with the regulation of trophoblast invasion are revealed through our RNA-seq database analysis. Notably, the genes negatively regulating trophoblast invasion, highlighted in red, were highly expressed in MD-MSCs under cAMP/MPA treatment (Fig. 3C and Supplementary Data 2). Additionally, we observed downregulation of Timp1, Timp3, and Timp4 in E2/P4-treated De-PAs, while Il33, Mmp12 and Mmp28 are upregulated (Fig. 3C-a). The protein-protein interactions were analyzed by STRING for these proteins, encoded by these genes, exhibit a high density of interactions involved in regulating trophoblast invasion. Particularly, tissue inhibitors of metalloproteinases (TIMPs) and matrix metalloproteinases (MMPs) exhibit a high level of evidenced known interactions (Fig. 3C-b). These analyses suggest that the innate regulatory mechanisms governing trophoblast invasion through E2/P4 decidualization in PAs differ from those observed in normal MD-MSCs. However, the differential expressions of these genes were not sufficient to account for the abnormal trophoblast invasion. Further research is necessary to fully understand the underlying mechanisms contributing to PAS. Particular attention should be given to dNK cells, due to their pivotal role in pregnancy through their intrinsic interactions with decidual cells and trophoblasts within the physiological microenvironment.

The induction of dNK-like cells from pNK cells is potentially achievable through a MD-MSC-based co-culture system.

It is well-established that, apart from decidual cells, dNK cells also play a crucial role in blastocyst implantation and placental formation. Due to limitations in acquiring dNK cells, we attempt to facilitate the in vitro conversion of dNK cells from pNK cells through co-culturing with MD-MSCs or De-MD-MSCs. The primary indicator for the successful conversion of dNK cells is primarily the reduced cytotoxicity characteristic of dNK cells. The timeline of the dNK-like cell conversion and the assessments are depicted in Fig. 4A. As the results showed, after co-culturing with MD-MSCs or De-MD-MSCs, the cytotoxicity of NK cells on trophoblasts decreased at each ratio of effector cells and target cells compared with pNK cells (Fig. 4B). Particularly in the dNK cell batches, dNK2 and dNK3 cells, the cytolytic ability was dramatically reduced by about 20–30% compared with pNK cells at the cell number ratio of 4:1 (Fig. 4B).

To exclude the possibility that the decline in NK cell cytotoxicity is not attributable to functional loss post-co-culture MD-MSCs, we further investigated the up-regulated expression levels of KIR2DL4, specifically expressed on dNK cell surfaces, to bind with HLA-G on trophoblast surfaces and activate the inhibitory signal contributing to immunotolerance to prevent trophoblasts from being cytolyzed. Firstly, we measured the levels of HLA-G on trophoblasts to confirm its presence (Fig. S3A). As anticipated, KIR2DL4 expression was notably higher, particularly on dNK cell batches, dNK2 and dNK3 cells, with percentages reaching 36.4% and 44.3% positive compared to the IgG control (Fig. 4C-a). The quantified statistics, normalized with pNK cells, demonstrated a dramatic increase in the expression levels of KIR2DL4 after co-culturing with trophoblasts (Fig. 4C-b and S3B). The up-regulated expression level of KIR2DL4 was consistent with the reduced cytotoxic ability. Apart from the inhibitory receptor expression, the cytokines and chemokines associated with immunotolerance secreted by decidual cells are found in the RNA-seq database, including Csf3, Il1β, and Tgfb1 (Fig. 4D-a and Supplementary Data 3). The protein-protein interactions analyzed by STRING for these 6 secreted proteins, exhibit interactions in regulating immune tolerance for dNK cells. Of noteworthy significance is the interaction between IL1β and CSF3, which is strongly supported by evidence from curated databases (Fig. 4D-b). These results suggest that MD-MSCs can potentially convert pNK cells into dNK-like cells and potentially mimic the immunotolerance on trophoblasts in vitro.

Identification of highly proliferated dNK-like cells in PAs

Following the successful induction of dNK-like cells in vitro, we conducted further investigation to examine the differential effects of MD-MSCs and PAs on dNK-like cells, in an attempt to find the potential causes of PAS. Initially, we assessed NK cell proliferation under co-culture conditions with MD-MSCs or PAs following various decidualization inductions (Fig. 5A). The experimental timeline spanned 10 days, with day 7 corresponding to the dNK-like cell conversion period. In the NK cell proliferation assay, we made an intriguing observation: PAs demonstrated a greater capacity to enhance NK cell proliferation compared to normal MD-MSCs, particularly in the E2/P4-treated groups (Fig. 5B-a). The quantified analysis clearly showed a three-fold increase in NK cell proliferation after co-culture with E2/P4-treated PAs compared to E2/P4-treated MD-MSCs on day 10 (Fig. 5B-b). This observation prompted us to explore the relationship between NK cell proliferation and trophoblast invasion further. Additionally, our RNA-seq analysis identified cytokines, known as key regulators of NK cell proliferation, which were predominantly upregulated in the PAs groups. Significantly, five genes potentially enhancing NK cell proliferation in E2/P4-treated De-PAs were annotated, including Cxcl12, Il33, Vegfa, Vegfc, and Tgfb1 (Fig. 5C-a and Supplementary Data 4), with the predicted interactions of these secreted proteins shown in Fig. 5C-b. VEGFA was not displayed in the interaction panel due to the absence of information in the STRING database. These findings suggested a potential association between abnormal NK cell proliferation and the development of PAS. Consequently, we propose the hypothesis that "Abnormal dNK cell proliferation may represent a significant contributing factor to the pathogenesis of PAS." However, further research is required to fully elucidate the underlying mechanisms and validate this hypothesis.

Increased dNK cell numbers may represent one of the key factors contributing to PAS.

To assess the earlier presumption that PAS could be attributed to dysregulated trophoblast invasion, we modified the previously established co-culture system to investigate the distinct impact of increasing the dNK cell batch dNK2 cell numbers on trophoblast invasion. The experimental timeline for this study is depicted in Fig. 6A. Given the complex interplay among the cells involved, we simplified the co-culture system by utilizing a dNK2 cell-conditioned medium. We anticipated that dNK2 cells would exhibit an enhanced capacity to promote trophoblast invasion with a dose-dependent correlation (Fig. 6B). Furthermore, dNK2 cells induced by PAs demonstrated a greater capacity to enhance trophoblast invasion compared to those induced by MD-MSCs (Fig. 6B). Statistical analysis was performed by normalizing the results with the control trophoblast alone group. This outcome indirectly suggests that increased dNK cell proliferation may be a pivotal factor in abnormal trophoblast invasion, potentially contributing to PAS as depicted in Fig. 7. The novel finding sheds light on the mechanisms underlying PAS and lays the groundwork for the development of treatments for this condition.

Previous studies have revealed the critical role that moderate trophoblast invasion plays in a successful pregnancy. Dysregulation of trophoblast invasion can lead to maternal lethal diseases, like the most severe pattern, PAS. PAS is associated with complicated interactions among decidual cells, trophoblasts, and dNK cells. The potential mechanisms of the PAS have not been clarified these days, due to the limitation of the cell derivation with ethical issues.

In the published studies, primary ESCs and even decidual cells were predominantly obtained from menstrual blood [31–32] or endometrial biopsies, including curettage [33–34] and elective termination of pregnancy samples [35–36]. However, these sources of ESCs have limitations regarding cell number and purity; and involve invasive surgical procedures that may cause discomfort to the subjects. Furthermore, obtaining specimens from normal women has proven particularly challenging due to ethical concerns. In this study, we have identified a distinct cell type among ESCs from normal placenta characterized by its potential for decidualization and high capacity for in vitro amplification. Biomarker investigations validated the decidualization process of MD-MSCs, with particular attention to recognized decidual cell markers such as Prl and Igfbp1 [37–38]. Our study observed a unique upregulation of Scara5 [26] in De-MD-MSCs, which has been suggested to play a pivotal role in crucial cellular functions during decidualization. Studies indicate that SCARA5 is downregulated in senescent decidual cells, suggesting its potential therapeutic implications in reproductive disorders [27], [39–40]. Further research is warranted to fully elucidate the mechanisms underlying the involvement of SCARA5 in decidualization and its implications for reproductive health. Given their unique in vitro decidualization potential and their ability to circumvent ethical issues, MD-MSCs stand out as a potentially valuable model for advancing the study of placental development and associated diseases.

In the trophoblast invasion assay, the validated EVT cell lines 3A-subE and HTR-8/SVneo were used in this study. We found that MD-MSCs treated with cAMP/MPA negatively regulate trophoblast invasion, in contrast to E2/P4-treated MD-MSCs, which enhance trophoblast invasion. The regulation of trophoblast invasion involves intricate cell-cell interactions and activation of multiple signaling pathways. Proteins secreted from decidual cells can either promote or inhibit trophoblast invasion, thus modulating trophoblast invasion levels. Gene expression analysis suggests that genes associated with trophoblast invasion enhancement (Mmps) are reduced, while genes associated with inhibiting trophoblast invasion (Timps) are upregulated in cAMP/MPA-treated MD-MSCs, consistent with their inhibitory effect on trophoblasts [41–45].

Building upon the well-established MD-MSC-based decidualization and trophoblast invasion system, we utilized pathological MD-MSCs to delve deeper into the underlying mechanisms of PAS. We observed diminished morphological changes and decreased expression levels of PRL and IGFBP1, both at mRNA and protein levels, in vitro and in vivo. Additionally, besides these two decidual markers, other annotated up-regulated genes under decidualization were found to be down-regulated under E2/P4 treatment. This observation may be due to abnormally high estrogen receptor expression, which has been associated with decidualization defects and endometriosis [46–47]. Regarding trophoblast invasion, no discernible effects on trophoblast invasion were observed between MD-MSCs and PAs. However, RNA-seq data revealed differential expression of molecules involved in trophoblast invasion regulation in E2/P4 treated De-PAs, notably down-regulated Timp1, Timp3 and Timp4 [42], [48–49] and upregulated Il33 [50–51], Mmp1, Mmp12 and Mmp28 [52–54], which may promote trophoblast invasion. Based on these results, we propose two possible explanations. Firstly, it is conceivable that the drug potency of cAMP/MPA is too high to reveal the decidualization defect in PAs. Secondly, abnormal trophoblast invasion may not be directly attributed to the inherent properties of defective decidual cells.

To investigate the indirect effects on trophoblast invasion, dNK cells were included in the co-culture system to explore their interactions with decidual cells and trophoblasts. However, due to the challenge in deriving dNK cells, we opted for in vitro induction methods to address this issue. Instead of chemical induction using TGFβ and demethylation agents [55], we employed decidual cell induction methods to closely mimic the physiological microenvironment [56]. Surprisingly, De-MD-MSCs demonstrated the potential to induce dNK-like cells from pNK cells, albeit with reduced cytolytic ability against trophoblasts. At the maternal-fetal interface, dNK cells play a crucial role in promoting immune tolerance through specific interactions between KIRs on dNK cells and HLA expressed on trophoblasts [57]. Notably, KIR2DL4 on maternal dNK cells can interact with soluble forms and HLA-G on fetal trophoblast cell surfaces, potentially modulating immune responses and promoting tolerance to the semi-allogeneic fetus during pregnancy [8], [16]. This interaction is pivotal in regulating maternal immune responses and maintaining the integrity of the maternal-fetal interface. Interestingly, KIR2DL4 expression on NK cells significantly increased when co-cultured with both E2/P4 and cAMP/MPA-treated De-MD-MSCs. The observed reduction in immune response may be attributed to cytokines secreted by decidual cells, including Csf3, Il-1β, and Tgfb1 [58–59], as suggested by RNA-seq data. Furthermore, we made a novel observation regarding the abnormal interaction between PAs and dNK-like cells. PAs exhibited a significant capacity to enhance dNK-like cell proliferation. However, the underlying mechanisms driving this abnormal proliferation of dNK cells in PAS have yet to be fully elucidated. Only a few published studies proposed that the reduced proliferation of dNK cells may be one of the contributing factors to PAS [60]. Thus, further evidence is required to clarify the correlation between dNK cell proliferation and PAS.

Based on the observed decidualization defect and the highly proliferated dNK-like cells induced by PAs, several secreted proteins implicated in promoting dNK cell proliferation and correlating with trophoblast invasion are found to be upregulated and annotated in RNA-seq data from PAs compared to MD-MSCs. CXCL12 secreted by decidual cells can bind to CXCR4 receptors on dNK cells or trophoblasts, indirectly or directly inducing trophoblast differentiation, invasion, and spiral artery remodeling [42], [61–63]. In addition, IL33 has been found essential for regulating trophoblast invasion and placental development [50–51]. Notably, TGFβ1, which has been suggested in evidence to reduce NK cell proliferation [64–65], is downregulated in E2/P4-treated De-PAs. Furthermore, VEGFA and VEGFC, secreted by both decidual cells and dNK cells, not only enhance trophoblast invasion but also promote angiogenesis and spiral artery remodeling, potentially leading to significant bleeding in PAS [66–67]. The up and downregulated genes observed in our study suggest a positive correlation among dNK cell proliferation, trophoblast invasion, and spiral artery remodeling. Functional validation reveals that conditioned medium derived from dNK-cells induced by E2/P4-treated De-PAs has a greater capacity to enhance trophoblast invasion in a dose-dependent manner compared to dNK-like cells induced by De-MD-MSCs.

Thes findings provide new insights into the underlying mechanisms of PAS through the decidualization defect and subsequent abnormal interactions among decidual cells, dNK cells, and trophoblasts. MD-MSCs have the potential to break the limitations of in vitro studies related to complex placental diseases and may significantly contribute to advancements in human health. However, the MD-MSCs-based co-culture model for studying placental disorders still presents challenges that require further efforts to overcome. The physiological identity of MD-MSCs in the endometrium requires further evidence to substantiate their correlation with decidualization and their role in placental formation, thereby highlighting the importance of this cell type. Due to a small clinical sample size, our results need further validation to ensure consistent findings through both in vitro cell assays and in vivo mouse models. The MD-MSC-based co-culture system needs to be augmented by co-culturing with various cell types to better mimic the complex microenvironment. Additionally, single-cell RNA-seq and cytokine array analyses should be conducted to validate the aforementioned results. We anticipate identifying specific molecular pathways to comprehensively elucidate the mechanisms underlying complex PAS in future research endeavors.

This study highlights the development of an MD-MSC-based system for in vitro investigation of the intricate interactions among decidual cells, trophoblasts, and dNK cell induction and their implications for PAS. Utilizing the MD-MSC-based co-culture system, decidualization defects characterized by decreased morphological changes and expression of decidual markers were observed in PAs. Additionally, MD-MSCs demonstrated the capacity to induce the differentiation of pNK cells into dNK-like cells, as evidenced by reduced cytotoxicity on trophoblasts and increased expression of KIR2DL4. Furthermore, PAs exhibited a high capacity to induce dNK-like cell proliferation, indirectly enhancing trophoblast invasion and spiral artery remodeling, possibly through the secretion of CXCL12, TGFβ1, VEGFA, and VEGFC. These findings provide new insights and lay the groundwork for further elucidating the complex pathophysiology of PAS through the novel MD-MSC cell type.

PAS	Placenta accreta spectrum
IUGR	Intrauterine growth restriction
EVT	Extravillous trophoblast
ESC	Endometrial stromal cell
dNK	Decidual natural killer cell
MD-MSC	Human placental maternal decidua-mesenchymal stem cell
pNK	Peripheral blood-derived natural killer cell
KIR	Killer Ig-like Receptor
HLA	Human leukocyte antigen
PRL	Prolactin
IGFBP1	Insulin-like growth factor binding protein 1
SCARA5	Scavenger receptor class A type 5
PBMC	Peripheral blood-derived mononuclear cell
ER	Estrogen receptor
PR	Progesterone receptor
E2	Estradiol
P4	Progesterone
cAMP	Cyclic adenosine monophosphate
MPA	Medroxyprogesterone acetate
BMMSC	Bone marrow-derived mesenchymal stem/stromal cell
PA	pathological MD-MSC from PAS patient
MMP	Matrix metalloproteinase
TIMP	Tissue inhibitors of metalloproteinase

Ethics approval and consent to participate.

The human placentas and blood were conducted according to the Institutional Review Board (IRB) guidelines issued by the National Taiwan University College of Medicine (IRB Approval No: 202308101RINC, No: 202312090RINC).

Consent for publication

Not applicable.

Competing interests

The authors declare no potential conflicts of interest.

Author details

¹Graduate Institute of Pharmacology, College of Medicine, National Taiwan University, Taipei 10051, Taiwan. ²Department of Obstetrics and Gynecology, National Taiwan University Hospital, Taipei 10041, Taiwan. ³VitaSpring Biomedical Co. Ltd., California 92618, USA. ⁴MediDiamond Inc., Taipei 11503, Taiwan. ⁵LuminX Biotech Co. Ltd., Taipei 11503, Taiwan.

Conflicts of interest:

The authors declared no potential conflicts of interest to the research, authorship, and/or publication of this article.

Funding

This work was supported by grant 110-2320-B-002-063-MY3 from the National Science and Technology Council to T.Y. Ling.

Author Contribution

Y.Z. Liu: Conceptualization, data curation, validation, investigation, writing, and editing.J.C. Shih: Conceptualization, data curation, validation, and investigation.M.S. Wu: Methodology, validation, and investigation.T.Y. Ling: Conceptualization, and funding acquisition.H.H. Lin: Formal analysis, validation, investigation, writing review, and editing.

Acknowledgement

Material and technical support: Placentas of PAS patients from Dr. Jing Chung Shih, Department of Obstetrics and Gynecology, National Taiwan University Hospital, Taipei, Taiwan. NK cell expansion kit (DSNK) from BIOMAB, INC., Innovation Incubation Center of National Taiwan University, Taipei, Taiwan. Flow cytometry analysis was provided by Flow Cytometric Analyzing and Sorting Core of the First Core Laboratory, National Taiwan University. Tissue section preparation was facilitated by the Laboratory Animal Center, College of Medicine, National Taiwan University.

Data Availability

All data supporting the findings of this study are available within the paper and its Supplementary Information.

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Dysregulation of Decidual NK Cell Proliferation by Impaired Decidual Cells: A Potential Contributor to Excessive Trophoblast Invasion in Placenta Accreta Spectrum

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Version 1

Abstract

Figures

Introduction

Materials and Methods

MD-MSCs and PAs isolation and culture

Decidual cell morphology quantification

Real-time PCR (RT-PCR)

Enzyme-linked immunosorbent assay (ELISA)

Immunohistochemistry (IHC) in tissue sections

Surface marker expression levels measured by flow cytometry

Trophoblast invasion assay

Peripheral blood mononuclear cells (PBMCs) isolation and peripheral blood natural killer (pNK) cells purification

Cytotoxicity assay for pNK cell and dNK-like cells

dNK-like cell proliferation assay

RNA-sequencing and data analysis

Statistical analysis

Results

Decidualization defects in PAs have been observed in the MD-MSC-based decidualization system

Identification of highly proliferated dNK-like cells in PAs

Discussion

Conclusions

Abbreviations

Declarations

Author details

Conflicts of interest:

Funding

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Supplementary Files

Status:

Version 1