Mesenchymal stem cells reside in a vascular niche in the decidua basalis and are absent in remodelled spiral arterioles
Introduction
The decidua basalis is a thin plate of maternal endometrial tissue that is structurally and functionally transformed by pregnancy and forms the attachment site of the placenta. During delivery, the decidua basalis adhering to the placenta is torn away from the underlying maternal tissues and remains attached to the placenta.
The decidua basalis and human placenta are rich sources of maternal and fetal derived mesenchymal stem cells (MSCs), respectively [1], [2], [3]. These cells are also referred to as mesenchymal stromal cells. Unlike placental MSCs, which are well characterised in vitro and are already in preclinical studies and clinical trials [4], [5], [6], [7], the in vitro properties and potential clinical applications of maternal decidua basalis MSCs (DMSCs) are less well known. Most important to this work is that our understanding of the in vivo function(s) of DMSCs is rudimentary. Identifying the stem cell niche can provide important clues as to the in vivo function(s) of DMSCs.
The stem cell niche is defined as the specialised local microenvironment where stem cells reside and the niche contributes directly toward stem cell maintenance [8]. Identifying the MSC niche in tissues is fundamental to understanding MSC function but locating the niche in human tissues is challenging. The retention of bromodeoxyuridine label by slow-cycling MSCs has been widely utilized to locate these cells in vivo [8], [9], [10]. MSC niches in bone marrow, periodontal ligament, dental pulp, and human adipose tissue were identified by employing a combination of characteristic cell surface markers [11], [12], [13], [14], [15], [16]. Combinations of markers are essential since no single, specific marker for MSCs has been identified. This probably reflects the heterogeneity of MSC populations in vivo, which adds further complexity to assigning the niche [17]. Some markers used to identify MSCs in a vascular niche are expressed in smooth muscle cells (α-smooth muscle actin/α-SMA), and pericytes (CD146, STRO-1, 3G5, frizzled-9/FZD-9). Using a combination of markers (CD146, VLA-1/CD49a, STRO-1, and 3G5), we localised fetal MSCs to a vascular niche within the chorionic villi of the placenta [18], consistent with vascular niches described for MSCs in many other organs and tissues [17].
The decidua basalis plays a critical role in human pregnancy, but the niche for MSCs in this tissue has not yet been identified. Early in human pregnancy (8–10 weeks), specialised invasive fetal extravillous trophoblast cells (EVTs) detach from the tips of fetal chorionic villi, invade into the decidua basalis and underlying myometrium, and migrate along the spiral arteries. EVTs infiltrate and replace, completely or partially, the vascular smooth muscle cells surrounding the spiral arterioles, and the endothelial cells that line the lumen of the spiral arterioles. This process remodels the spiral arterioles into large diameter conduit vessels of low resistance, which facilitates increased blood flow to the intervillous space of the placenta allowing it to meet the increasing nutritional demands of the rapidly growing fetus. Thus, remodelling of maternal spiral arterioles is essential for successful pregnancy [19], [20]. Identification of the DMSC niche may provide important clues as to the role DMSCs play in placental development, and specifically in spiral arteriole remodelling.
In this study, we employed a strategy similar to our previous studies on placental MSC niches [18], [21]. First, DMSCs were isolated from the decidua basalis according to published methods [22], [23], [24]. Next, the cultured cells were assessed for MSC criteria proposed by the International Society of Cellular Therapy (ISCT) [25]. These criteria include that MSCs must adhere to plastic surfaces, should express CD105, CD73 and CD90 but not CD34, CD14, CD19, CD11b, CD79α or HLA-DR, and have multipotent differentiation potential [25]. Further criteria set by Parolini et al. for placental MSCs include the determination of the fetal or maternal origin of the cells [26]. Then, immunocytochemistry was performed on cultured DMSCs to determine whether they were positive for markers used to define MSC niches (FZD-9, 3G5, α-SMA, and STRO-1). Finally, multi-label immunofluorescence staining of placental bed biopsies with this combination of niche markers was used to identify the DMSC niche in the decidua basalis before and after spiral arteriole remodelling.
Section snippets
Tissue collection
Placental samples were collected from healthy women with medically uncomplicated pregnancies following Caesarean section or vaginal delivery at term (n = 5). The tissue had the typical morphology of a healthy placenta with no obvious signs of calcification, infarcts or meconium staining. Exclusion criteria for tissue collection were twin or triplet pregnancy, maternal smoking, drug dependency, intrauterine infection, prolonged rupture of the fetal membranes, and placental abruption. Informed
Isolation and characterization of DMSCs
Cells were isolated from the decidua basalis attached to villous placental tissue collected from women with uncomplicated pregnancies delivered by Caesarean section or vaginal delivery at term. The cells adhered to tissue culture flasks and had the fibroblast-like appearance typical of MSCs (Fig. 1A). The yield of DMSCs at P0 was not determined because the cell population was not homogeneous. At P1, when the population displayed a homogeneous, fibroblast-like morphology, the yield was 5 × 105
Discussion
Cells were successfully isolated from the decidua basalis that remains attached to the placenta following delivery, using a tissue mincing and enzymatic digestion method. Following in vitro growth and passaging, the cells appeared homogeneous and exhibited the characteristic fibroblastic morphology of MSCs (Fig. 1Ai). Cells prepared met accepted ISCT criteria for MSCs, including adherence to plastic cultureware, surface marker expression, and differentiation potential [25].
Immunophenotyping
Conflict of interest
The author(s) declared that there is no potential conflict of interest with respect to the research, authorship, and/or publication of this article.
Acknowledgements
We acknowledge the patients who consented to provide their samples, the obstetric staff who provided the placental bed biopsy samples, and the clinical research midwives at the Royal Women's Hospital, Sue Duggan and Moira Stewart for sample collection. We thank Melissa Duggan, Dr. Maria Kokkinos, and May Grgurinovic for their excellent technical assistance. This work was supported by grant funds from King Abdullah International Medical Research Centre (Grant No. RC08/114), the Royal Women's
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