P-349 Modelling the impact of acute and chronic decidual senescence on endometrial stemness in 3D assembloids

Abstract

Abstract Study question Is de-differentiation of committed endometrial stromal and epithelial cells in response to decidual senescence involved in regulating stemness of cycling human endometrium? Summary answer Transient decidual senescence promotes endometrial tissue rejuvenation by reprogramming stromal and epithelial cells into progenitor stem-like cells whereas chronic senescence causes stem cell depletion. What is known already In wound healing, acute but not prolonged senescence, a cellular state characterised by permanent cell cycle arrest and production of a complex secretome rich in inflammatory mediators, ECM proteins and proteinases, growth factors and angiogenic modulators, has been shown to promote de-differentiation of committed cells into stem-like progenitors, thereby enhancing tissue regeneration upon immune clearance of senescent cells. Menstrual repair depends on endometrial progenitor cells but whether they represent stromal and epithelial cells that have de-differentiated in response to acute premenstrual decidual senescence is not known. Study design, size, duration Endometrial 'instant' assembloids, consisting of gland organoids and primary stromal cells in collagen hydrogels, were established from freshly isolated cells from mid-luteal endometrial biopsies. The assembloids were then subjected over 36 days to 4 cycles of decidualization, a process associated with acute inflammatory senescence, followed by hormonal withdrawal. To induce chronic senescence, assembloids were decidualized continuously for 14 days. Stemness of assembloids was assessed in undifferentiated and decidualized cultures at the end of each ‘cycle’. Participants/materials, setting, methods 'Instant' assembloids, which closely recapitulate native endometrium, were established in collagen hydrogels from 10 midluteal biopsies and subjected to cyclical or prolonged decidualization. Decidualization was monitored by RT-qPCR analysis, using epithelial and stromal cells isolated at regular timepoints. The level of stemness of stromal and epithelial cells was measured by colony-forming unit (CFU) and organoid formation efficacy (OFE) assays, respectively. Main results and the role of chance Repeated cycles of hormonal stimulation and withdrawal resulted in cyclical decidualization of instant assembloids, as characterised by the induction of decidual stromal (PRL, SCARA5 and DIO2) and epithelial (PAEP and SPP1) marker genes when compared to parallel undifferentiated assembloids. Cyclicity enabled cells to maintain a healthy state and preserved the structural integrity of the assembloids. Further, cyclical decidualization of assembloids enhanced CFU activity and OFE activity of stromal and epithelial cells, respectively, indicative of active de-differentiation of committed cells. By contrast, chronic senescence, as induced by a prolonged decidualization, resulted in stem cell depletion in both glandular and stromal compartments and progressive loss of structural integrity of assembloids. We observed a reduced expression of the decidual marker gene PRL and an enhanced expression of IGFBP1 representing the increased stress state of cells. Taken together, induction of acute decidual senescence resulted in a robust de-differentiation response and increased abundance of stromal and epithelial progenitor cells, whereas prolonged senescence caused stem cell exhaustion in both cellular compartments. Limitations, reasons for caution Although the cellular responses observed in our ‘instant’ assembloid model were robust, caution is warranted when extrapolating from in vitro observations. Further, although the ‘instant’ assembloid model enables co-culturing of endometrial endothelial and immune cells, current hydrogels greatly limit their migratory capacity. The mechanisms of cellular de-differentiation are incompletely understood. Wider implications of the findings Our findings indicate that the level of premenstrual decidual senescence in the superficial layer regulates stemness in the basal layer, thus ensuring inter-cycle endometrial homeostasis. Conversely, prolonged decidual senescence associated with clinical miscarriages may plausibly increase the risk of further pregnancy loss by depleting stemness of the regenerative basal layer. Trial registration number N/A