Endometrial regeneration : unraveling the mysteries of the stroma

Abstract: The healthy endometrium has the unique capacity of shedding and regenerating its functional layer once every menstrual cycle, under the regulation of hormones, progenitor cell populations and inflammatory mediators. Thanks to this meticulously timed cycle the uterus is regularly renewed in preparation for blastocyst implantation. However, if any of these factors are perturbed, the result may be endometrial disorders with intrauterine scar formation, dysregulated proliferation and heavy menstrual bleeding. This PhD project addresses the interplay of these factors specifically within the endometrial stromal compartment in ensuring healthy endometrial regeneration and homeostasis, thereby investigating future therapeutic targets for benign gynecological disorders. Cells and tissue from endometrial biopsies and bone marrow aspirate were studied using multiple cellular and molecular techniques. Stromal cells have been characterized in terms of their phenotype, their transcriptome and their ability to immunomodulate, providing a starting point for future endometrial stromal cell therapy development. In study I, the transcriptional profile of progesterone receptor modulator associated endometrial changes (PAEC) was studied in women with three months of continuous mifepristone treatment to understand the future implications of PAEC and the safety of longterm mifepristone use. Our microarray findings indicate that progesterone withdrawal and unopposed estrogen surge alter the endometrial structural organization and extracellular matrix composition, particularly affecting the stromal compartment in the tissue. No differentially regulated genes were involved in endometrial-cancer associated pathways. In study II, endometrial stromal cells (eSCs) were characterized in terms of their phenotype, immunomodulation and tumorigenicity for early pre-clinical cell therapy development. eSCs demonstrated a mesenchymal stromal cell (MSC) surface marker profile and multipotency, while retaining chromosomal stability and showing no tumorigenicity after in vitro expansion. When stimulated with pro-inflammatory cytokines, eSCs presented with an antiinflammatory phenotype and secreted immunomodulatory factors, but did not express human leukocyte antigen class II on their cell surface. eSCs suppressed CD4+ T cell proliferation and activation while significantly modulating their differentiation state, upregulating CD4+ effector memory T cells. The cellular diversity within the endometrial functional layer’s stromal compartment was explored in study III using single cell RNA sequencing. Different computational tools and an external dataset were used for analysis and validation. The study revealed diverse stromal subsets with transcriptional profiles representing different stromal activation states and niches important in wound healing, regeneration and immunomodulation. Several of these stromal transcriptional profiles could be validated in placenta derived decidua, suggesting the transcriptional profiles can withstand cycle changes and placentation. In study IV we determined the effects of blood exposure on bone marrow (BM) MSC viability and immunomodulatory functions, exposing BM MSCs to human blood products in vitro and evaluating their interactions with complement and the peripheral immune repertoire. Plasma exposure induced lysis of BM MSCs, while surviving BM MSCs had C3c bound to their surface. The MSC secretome reduced monocyte number and recruitment with many remaining monocytes skewed towards a classical, anti-inflammatory phenotype. Frequencies of immune modulating myeloid derived suppressor cells (MDSCs), both monocytic and polymorphonuclear, were also increased in response to BM MSCs. These data indicate that MSCs rapidly die once exposed to blood, but can still exert an anti-inflammatory response through the skewing of monocytes and upregulation of MDSCs. We hypothesize that this shift in the peripheral repertoire indirectly regulates adaptive immune cells’ response to the long-term tolerogenic effect seen after MSC intravenous infusion. In summary, endometrial regeneration is tightly regulated by hormones, immune cells and their interactions with stromal cells. Our research has provided new, detailed information on the stromal compartment by demonstrating there are multiple stromal subsets with different transcriptional profiles, presumably fulfilling multiple roles. Furthermore, eSCs have the ability to respond to inflammatory cues, as well as directly and indirectly modulating immune cells through their secretome. Additionally, BM MSCs exposed to blood can modulate the innate immune response even after cell death due to their release of soluble factors which induce an anti-inflammatory monocyte phenotype which lasts beyond their clearance from circulation. We conclude that stromal cells from different tissues have exceptional regulatory capacities in tissue homeostasis, inflammation and healing.