The role of decidual mesenchymal stem/stromal cells and their secreted extracellular vesicles in ageing of the human placenta

Abstract

Ageing is an inevitable process associated with age-related diseases. Unhealthy ageing is a major risk factor for many chronic diseases including cardiovascular and neurodegenerative diseases. The human placenta, despite being a short-lived and transient organ, does not escape the effects of ageing and aberrant placental ageing is a feature of important placental pathologies including preeclampsia (PE) and fetal growth restriction (FGR). Recent evidence shows placental tissues undergo significant molecular age-related changes in a short period, between early-term (ET) (37 0/7– 38 6/7 weeks gestation) to late/post-term (L/PT) (41 0/7 weeks gestation and beyond), after the due date of the pregnancy has been reached. Moreover, there is a dramatic increase in the risk of unexplained antepartum stillbirths in pregnancies that progress into the L/PT period, which may be linked to increased ageing of placenta. The role of ageing and the underlying mechanisms that regulate aberrant placental ageing during this period remain unclear.

The maternal-fetal interface develops between the chorionic placenta to the maternal uterine wall and is vital for the maintenance of a healthy pregnancy. The decidua is the maternal tissue between the placenta and the muscular uterine wall. Age-related tissue and cellular changes are evident throughout the decidua and placenta. The decidua basalis tissue, which is the focus of this study, and underlying myometrium are continually exposed to damaging reactive oxygen species (ROS) and inflammatory molecules secreted primarily by the placenta. The decidua basalis must withstand harsh physical conditions associated with changes in blood flow into the intervillous space during uncomplicated pregnancy. However, the extent of decidual ageing in uncomplicated pregnancies that reach term, and how decidual ageing affects the placenta, are not well understood.

Stem cell exhaustion and dysfunction are major contributors to the decline in tissue and organ functionality associated with ageing. The placenta and decidua are rich sources of stem cells, particularly mesenchymal stem/stromal cells (MSCs) which are highly active and involved in placental development and growth. However, the role of stem cells and their association with ageing of the chorionic placenta and maternal decidua basalis, especially in the short period between ET and L/PT, has not been investigated. The work of this thesis focuses on maternal decidua basalis derived MSCs (DMSCs).

The first aim of this study was to compare ageing/senescence functions of ET DMSCs (ET- DMSCs) from (37-39 gestational weeks) with L/PT DMSCs (L/PT-DMSCs) (41-42 gestational weeks). The second aim was to screen for differentially expressed known and novel small RNAs between ET-DMSCs and L/PT-DMSCs. The third aim was to analyse and characterise ET-DMSCs and L/PT-DMSCs secreted extracellular vesicles (EVs). EVs are nanosized particles secreted by MSCs, and they potentially regulate health and the ageing processes. The final aim of this work was to treat ET-DMSCs, L/PT-DMSCs and PE-DMSCs with a low dose of aspirin, which is an anti-aging and anti-inflammatory drug.

ET, L/PT, and PE-affected placentae were collected with informed consent. DMSCs were successfully isolated from decidua basalis of ET, L/PT, and PE placentae using an enzymatic digestion method and then characterised for their stem/stromal cell properties. The phenotypic characterization of DMSCs met the minimum criteria of MSCs.

L/PT-DMSCs, when compared to ET-DMSCs, showed significantly lower cell proliferation and a significant higher level of cell apoptosis. Using a quantitative Aldefluor assay, ET-DMSCs showed a significantly higher resistance to oxidative stress compared with L/PT-DMSCs. There was a significant decrease in antioxidant capacity of L/PT-DMSCs compared with ET-DMSCs. Western blot analysis revealed increased expression of the stress-mediated p38MAPK protein in L/PT- DMSCs. These data provide the first evidence of advanced ageing and loss of important stem cell functions in L/PT-DMSCs.

Sequencing of small RNAs and validation by qRT-PCR demonstrated miR-516-5p, which is a member of the chromosome 19 miRNA cluster (C19MC), was present at significantly lower levels in L/PT-DMSCs. This miRNA was also contained in EVs isolated from ET- and L/PT-DMSCs and the difference in levels of this miRNA in whole ET- and L/PT-DMSCs was also maintained in their respective EVs. This demonstrated that EV miRNAs are origin-dependent and their differences in levels reflected those in the ET- and L/PT-DMSCs that they were secreted from. EVs from both groups were further analysed using a novel method resonance enhanced atomic force microscopy and infrared spectroscopy (AFM-IR) which revealed a distinct fingerprint of biomolecules between these two groups. There were substantial differences in peak intensity of EVs contents between ET- and L/PT-DMSCs, particularly those associated with lipid and protein content. The combination of conventional assays and AFM-IR provide the first evidence of advanced age-related, and other changes, in EVs from L/PT decidua MSCs.

Finally, low dose aspirin treatment improved PE-DMSCs functionality by a novel mechanism via increasing cell adhesion. Aspirin treatment also increased antioxidant capacity and decreased inflammatory markers in PE-DMSCs. Aspirin had no similar effect on ET- and L/PT-DMSCs.