Preeclampsia is a pregnancy-specific disorder defined by the new onset of hypertension and proteinuria after 20 weeks of gestation. Although its precise etiology is still unknown, there is evidence suggesting that it may be a consequence of impaired decidual and stromal cell function. Recently, a stem cell population derived from endometrial tissue and isolated from menstrual effluent called menstrual stem cells (MenSCs) has been identified. MenSCs exhibit important angiogenic and inflammatory properties that may contribute to both normal and pathological complications of implantation and placentation, including preeclampsia. We hypothesized that the angiogenic and inflammatory activity of MenSCs is altered in women who have a past history of preeclampsia and that this phenotype persists postpartum. The primary outcome measures were stromal progenitor cell formation,
Preeclampsia (PE) is a life-threatening vascular disorder of pregnancy due to a failing placenta that affects 3% to 5% of all pregnancies and is a major cause of maternal and perinatal morbidity and mortality [
Recently, endometrial cells have demonstrated to play a central role in both normal and abnormal early pregnancy development [
In the past decade, a population of mesenchymal stem cells (MSCs) with endometrial origin has been identified and characterized from human endometrial tissue and menstrual fluid [
Although implantation and angiogenesis are both impaired in PE, the relation between the angiogenic properties of MSCs derived from the endometrium and the pathology of PE has not been evaluated. In this study, we characterized the phenotype of MenSCs isolated from women who previously developed PE (MenSCs-PE) in comparison with MenSCs isolated from healthy donors (MenSCs-C) in relation to their morphological, immunophenotypic, and functional characteristics. In a second phase, we evaluated the angiogenic properties of these cells by measuring their capacity of tube formation and secretion of the soluble factors, VEGF and endoglin, and cytokines that can modulate angiogenesis such as IL-6, IL-1
All human samples and cells used in this study were collected and harvested with the informed consent of the donor as approved by the ethical scientific committee of Clinica Davila and Universidad de los Andes. MenSCs were obtained from nine healthy donors (control) and six women who had PE on their previous pregnancies (9-12 months postpartum). Demographic characteristics were recorded. All women were using natural family planning, were not breastfeeding, had regular menses, and had not used contraceptives for at least 3 months before enrolment in the study.
MenSCs were isolated as described previously [
For phenotypic characterization, cells were harvested, washed with cytometer buffer (PBS + 0.2% BSA + 0.01% sodium azide (Sigma-Aldrich, MI, USA)), and incubated with the specific labelled antibodies in cytometer buffer for 20 min at 4°C. Antibodies for human cell surface antigens CD14, CD44, CD90, CD105, CD73, CD45, CD34, HLA-ABC, HLA-DR, and CD146 were purchased from BD Biosciences, MD, USA, R&D Systems, MN, USA, and BioLegend, CA, USA. Matching isotype antibodies were used as negative controls in all experiments. In addition, LIVE/DEAD® Fixable dead cell stain kit (Invitrogen, CA, USA) was used to determine the viability of cells by flow cytometry according to the manufacturer’s protocol. Data were collected using a fluorescence-activated cell sorting (FACS) Canto II flow cytometer (BD Biosciences) and analyzed on the FlowJo analysis software.
MenSCs were induced to differentiation into chondrogenic, osteogenic, and adipogenic lineages using specific media over 21 days. For osteogenic differentiation, media were composed of DMEM high glucose (Corning, MA, USA) supplemented with FBS 5% (Gibco), 155 nM ascorbic acid (Sigma), 10
Tri-differentiation primers.
Gene | Lineage | Primer |
---|---|---|
Osteogenic | CGG AAT GCC TCT GCT GTT AT | |
Osteogenic | TTC CCG AGG TCC ATC TAC TG | |
Osteogenic | GGC GCT ACC TGT ATC AAT GG | |
Osteogenic | TCA GCC AAC TCG TCA CAG TC | |
Adipogenic | ATG GGA TGG AAA ATC AAC CA | |
Adipogenic | GTG GAA GTG ACG CCT TTC AT | |
Adipogenic | ATG GAG TCC ACG AGA TCA TT | |
Adipogenic | CGC AGG CTC TTT AGA AAC TC | |
Housekeeping | CAG CCT CAA GAT CAT CAG CA | |
Housekeeping | CAT GAG TCC TTC CAC GAT AC |
To quantify the frequency of stromal progenitors, cells obtained after Ficoll centrifugation of the menstrual effluent were plated at a density of 100, 1,000, 10,000, and 100,000 nucleated cells/cm2 in DMEM. The medium was changed the next day to remove nonadherent cells. The frequency of progenitors was calculated following the extreme limiting dilution analysis (ELDA) method for comparing depleted and enriched populations in stem cells [
MenSCs were cultured at 1,000 cells/cm2 in 24-well plates (Falcon, Corning) in complete DMEM (10% FBS, 1% penicillin-streptomycin). Cell proliferation and viability were determined at days 3, 6, and 9 by the SRB Assay (BioVision, CA, USA) and by spectrophotometric quantification (absorbance, 492 nm) according to the manufacturer’s instructions.
MenSCs were cultured under an endometrial hormonal milieu. Briefly, 120,000 MenSCs were cultured in a 6-well plate with complete DMEM without phenol red (Gibco) and exposed to 5% oxygen conditions (5% O2). 24 h after, the medium was replaced with DMEM without phenol red and 10% FBS, treated with active carbon and 1% penicillin-streptomycin, supplemented with estrogen (E2; 213 pg/ml, Sigma-Aldrich, MI, USA), and cultured at 5% O2 for 24 h. The medium was replaced for the same media described above but supplemented with both estrogen and progesterone (E2; 146 pg/ml, P4; 11 ng/ml, respectively, Sigma-Aldrich, MI, USA) and cultured at 5% O2. 24 h later, conditioned media were harvested, centrifuged at 500 g for 5 min, and stored at -80°C for subsequent analysis: angiogenesis assay, ELISA, and protein solution array assays (Luminex Corporation, TX, USA). MenSC cellular lysates were collected for Western blot analysis.
Tube formation in Matrigel (Corning) was used as a functional assay to evaluate the angiogenic properties of MenSCs. Briefly, human umbilical vein endothelial cells (HUVECs) were seeded in 48-well plates precoated with 70
Protein lysates were extracted from MenSCs exposed to endometrial conditions using RIPA buffer. Protein concentration was measured using the Qubit Protein Assay kit (Thermo Fisher Scientific), and 5
The conditioned medium harvested from MenSCs cultured under an endometrial hormonal microenvironment was concentrated using 10 kDa Amicon Ultra-4 filters (Millipore, St. Charles, MO, USA), and the protein concentration of the concentrated conditioned medium was measured using the Qubit Protein Assay kit (Thermo Fisher Scientific). VEGF, soluble endoglin (sEng), soluble fms-like tyrosine kinase-1 (sFlt1), angiopoietin 1, and angiopoietin 2 concentrations were detected using ELISA kits (VEGF, cat# DY293B; endoglin, cat# DY1097; sFlt1, cat# DVR100C; angiopoietin 1, cat# DANG10; and angiopoietin-2, cat# DANG20; R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol.
The secreted cytokine profile was determined using a commercial 11-multiplex fluorescent bead-based immunoassay (R&D Systems). The concentration of the following cytokines was determined: IL-8, IFN-
Statistical analyses were performed using the
The demographic characteristics of the PE (case group) and control groups are presented in Table
Demographic characteristics.
Characteristics | Control ( |
PE ( |
|
---|---|---|---|
Age (years), median (IQ) | 29.00 (28.00-33.00) | 30.50 (26.75-34.25) | 0.9319 |
Weight (kg), median (IQ) | 60.00 (53.50-68.00) | 73.50 (59.00-83.75) | 0.0939 |
Height (mt), median (IQ) | 1.650 (1.610-1.690) | 1.605 (1.573-1.658) | 0.2302 |
BMI (kg/cm2), median (IQ) | 22.00 (19.40-24.80) | 27.30 (23.18-33.68) | 0.0496∗ |
Nullipara, |
4 (44.4) | 0 (0) | |
Miscarriages, |
1 (11.1) | 1 (16.7) | |
Early PE, | 4 (66.7) | ||
Late PE, | 2 (33.3) | ||
Early PE (weeks), median (IQ) | 27.5 (24.5-27.5) | ||
Late PE, (weeks) median (IQ) | 37.1 (36.4-37.7) |
Abbreviations: kg: kilograms; mt: meters; BMI: body mass index (kilograms/square meters). Results are
For case and control groups, MenSC morphology, phenotypic characteristics, and potential to differentiate into adipocytes, osteoblasts, and chondrocytes were compared. Both cases and controls displayed similar fibroblast-like morphology (Figure
Morphology, phenotypic markers, and multilineage capacities of MenSCs-PE. (a) Fibroblast-like morphology of MenSCs isolated from healthy women (control) and women with a previous preeclampsia (PE). MenSCs from PE showed the same morphology as MenSCs isolated from control. (b) Phenotypic characterization of MSC surface markers in MenSCs isolated from women with a history of PE (red) and control (black) and their respective isotype control (gray); the cells were analyzed by flow cytometry. All MenSCs from both groups were positive for CD90, CD73, CD105 (endoglin), and CD44 and negative for CD14, CD34, HLA-DR, and CD45. (c) Osteogenic differentiation of MenSCs isolated from healthy control women and women who developed PE was evaluated by measuring mRNA expression of RUNX2 and osteocalcin by qPCR. (d) Adipogenic differentiation of MenSCs isolated from healthy control women and women who developed PE was evaluated by measuring FABP4 and PPAR-
To compare functional properties of MenSCs, the proliferative and clonogenic potential of these cells was assessed. The proliferation rate was similar in both case and control groups (Figure
Proliferative and clonogenic potential of MenSCs-PE. (a) Proliferative capacity of MenSCs isolated from healthy women (control) and women who had a past history of preeclampsia (PE). Cell proliferation was evaluated at days 3, 6, and 9 by the SRB assay. MenSCs-PE showed a similar proliferation rate when compared to MenSCs isolated from healthy women. (b) Stromal progenitor capacity; serial dilutions of a defined number of cells in passage 0 (P0) were cultured, and the potential to form stromal progenitors was evaluated. MenSCs-PE show less initial frequency of stromal progenitors than do MenSCs from control women. (c) CFU capacity; serial dilutions of a defined number of cells in passage 4 (P4) were cultured, and the potential to form CFU was evaluated. MenSCs-PE show less frequency of CFU capacity after cell expansion in low dilutions. (d) Representative images of CFU after cell expansion in low dilutions of MenSCs-PE and healthy women (control) cultured at P4. Results are the mean + SEM (standard error of the mean). Statistical analysis was performed using Student’s
Since the human uterus is exposed to oxygen tensions of 2-5% [
MenSCs isolated from women with a previous pregnancy complicated by PE have reduced angiogenic capacity compared to controls (Figure
Reduced tube formation capacity on endothelial cells under MenSCs-PE conditioned media
VEGF protein expression (Figures
Reduced expression and secretion of VEGF and reduced expression of endoglin on MenSCs-PE
Levels of angiogenic factors secreted from MenSCs.
Characteristics | Control ( |
PE ( |
|
---|---|---|---|
VEGF (pg/ml)/mg total protein, median (IQ) | 20.05 (13.70-45.18) | 11.40 (5.438-17.60) | 0.0233∗ |
sEng (ng/ml)/mg total protein, median (IQ) | 0.066 (0.042-0.082) | 0.088 (0.061-0.1845) | 0.0637 |
sFlt1 (pg/ml)/mg total protein, median (IQ) | 81.38 (30.43-117.2) | 91.36 (20.39-281.3) | 0.3826 |
Angiopoietin-1 (pg/ml)/mg total protein, median (IQ) | 103.3 (28.99-168.9) | 136.3 (48.26-239.7) | 0.2264 |
Angiopoietin-2 (pg/ml)/mg total protein, median (IQ) | 59.75 (40.76-128.9) | 61.34 (37.49-127.7) | 0.3275 |
Abbreviations: VEGF: vascular endothelial growth factor; sEng: soluble endoglin; sFlt1: soluble fms-like tyrosine kinase-1; pg: picograms; ng: nanograms; ml: milliliters; mg: milligrams. Results are
Increased secretion of IL-6 on MenSCs-PE. (a) IL-6, (b) IL-1
Levels of proinflammatory cytokines secreted from MenSCs.
Characteristics | Control ( |
PE ( |
|
---|---|---|---|
IL-8 (pg/ml)/mg total protein, median (IQ) | 1359 (424.2-1423) | 1355 (1126-1380) | 0.4645 |
IFN- |
4.913 (2.613-6.524) | 5.465 (3.825-7.933) | 0.2757 |
IL-1 |
2.857 (0.697-3.694) | 4.418 (3.862-5.020) | 0.0571 |
IL-12p70 (pg/ml)/mg total protein, median (IQ) | 7.911 (3.598-10.04) | 9.369 (5.741-12.16) | 0.2537 |
IL-2 (pg/ml)/mg total protein, median (IQ) | 6.623 (5.346-7.394) | 6.882 (4.991-9.218) | 0.3247 |
TNF- |
1.178 (0.389-1.699) | 1.296 (0.855-2.405) | 0.2428 |
GM-CSF (pg/ml)/mg total protein, median (IQ) | 17.55 (9.24-37.64) | 34.20 (22.45-130.3) | 0.0745 |
IL-1 |
2.402 (1.183-3.586) | 2.619 (1.581-5.668) | 0.3423 |
IL-10 (pg/ml)/mg total protein, median (IQ) | 0.336 (0.209-0.366) | 0.274 (0.220-0.375) | 0.4382 |
IL-17A (pg/ml)/mg total protein, median (IQ) | 1.095 (0.619-1.363) | 1.370 (0.753-1.630) | 0.2194 |
IL-6 (pg/ml)/mg total protein, median (IQ) | 228.6 (23.40-450.8) | 349.8 (299.8-1032) | 0.0145∗ |
Abbreviations: pg: picograms; ml: milliliters; mg: milligrams. Results are
Since decidualization has been related to the development of PE, we evaluated the mRNA expression of genes involved in the decidualization process, such as IGFBP-1 and TGF-
The menstrual fluid is generally considered to represent a combination of blood and desquamated cells from the endometrium, many of which are nonviable or in a senescent state. A subpopulation of these cells displays stem cell properties, including angiogenic activity. The data obtained in this study suggest that these cells retain phenotypic variations postpartum that may be associated with and account for the abnormal implantation process observed in preeclampsia. MenSCs obtained from women with a previous preeclamptic pregnancy express less VEGF and membrane-bound endoglin, induce less endothelial cell tube formation, and release more IL-6 than do cells obtained from women with a normotensive previous pregnancy or from women that were never pregnant. The observed phenotypic variations are constituent with the pathophysiology of PE.
Different studies have shown the importance of endometrial cells in the natural selection of human embryos in the process of implantation [
Decidualization induces a switch in the secretion of different secreted factors around the perivascular niche to promote trophoblast invasion and to regulate immune response. Decidualization defects have been described to be involved in the development of PE [
Venkatesha and collaborators showed that soluble endoglin (sEng) plays an important role in the pathogenesis of PE affecting the placental vascular function [
In addition to changes in Eng on MenSC-PE, we also observed an increased secretion of IL-6 from these cells. Indeed, PE has been associated with an immune imbalance which induces a systemic inflammation [
In summary, previous studies suggest that endometrial stromal cells obtained from individual patients closely phenocopy the decidualization process. Menstrual fluid contains a subpopulation of mesenchymal stem cells of endometrial origin. Menstrual stem cells from women who had a previous preeclampsia display different angiogenic and inflammatory properties than do those obtained from patients who had a previous normal pregnancy. These data may be useful in the development of predictive models to identify patients at risk of preeclampsia in the preconceptional stage.
The data used to support the findings of this study are included within the article and within the supplementary information file(s).
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Manuel Varas-Godoy and Stephanie Acuña-Gallardo contributed equally to this work.
This work was funded by Comisión Nacional de Investigación Científica y Tecnológica: Fondecyt 1140119 to Sebastian E. Illanes and Fondecyt 11150624 to Manuel Varas-Godoy. We thank the technical expertise and support of Alex Cabrera in the flow cytometry experiments and Pía Venegas for her valuable contribution to the sample collection and patient recruitment.
Supplementary Figures_Final: file with the legends of each Supplementary Figures. Supplementary Material_Final: file with all the methods related to the experiments performed for the Supplementary Figures. Supplementary Figure 1, 2, 3, 4, 5 and 6: supplementary figures in EPS format. The description of each figure is in the Supplementary Figures_Final file.