Generation of a three-dimensional collagen scaffold-based model of the human endometrium.

The endometrium is the secretory lining of the uterus that undergoes dynamic changes throughout the menstrual cycle in preparation for implantation and a pregnancy. Recently, endometrial organoids (EO) were established to study the glandular epithelium. We have built upon this advance and developed a multi-cellular model containing both endometrial stromal and epithelial cells. We use porous collagen scaffolds produced with controlled lyophilization to direct cellular organization, integrating organoids with primary isolates of stromal cells. The internal pore structure of the scaffold was optimized for stromal cell culture in a systematic study, finding an optimal average pore size of 101 µm. EO seeded organize to form a luminal-like epithelial layer, on the surface of the scaffold. The cells polarize with their apical surface carrying microvilli and cilia that face the pore cavities and their basal surface attaching to the scaffold with the formation of extracellular matrix proteins. Both cell types are hormone responsive on the scaffold, with hormone stimulation resulting in epithelial differentiation and stromal decidualization

Trophoblast organoids as a model for maternal-fetal interactions during human placentation.

The placenta is the extraembryonic organ that supports the fetus during intrauterine life. Although placental dysfunction results in major disorders of pregnancy with immediate and lifelong consequences for the mother and child, our knowledge of the human placenta is limited owing to a lack of functional experimental models1. After implantation, the trophectoderm of the blastocyst rapidly proliferates and generates the trophoblast, the unique cell type of the placenta. In vivo, proliferative villous cytotrophoblast cells differentiate into two main sub-populations: syncytiotrophoblast, the multinucleated epithelium of the villi responsible for nutrient exchange and hormone production, and extravillous trophoblast cells, which anchor the placenta to the maternal decidua and transform the maternal spiral arteries2. Here we describe the generation of long-term, genetically stable organoid cultures of trophoblast that can differentiate into both syncytiotrophoblast and extravillous trophoblast. We used human leukocyte antigen (HLA) typing to confirm that the organoids were derived from the fetus, and verified their identities against four trophoblast-specific criteria3. The cultures organize into villous-like structures, and we detected the secretion of placental-specific peptides and hormones, including human chorionic gonadotropin (hCG), growth differentiation factor 15 (GDF15) and pregnancy-specific glycoprotein (PSG) by mass spectrometry. The organoids also differentiate into HLA-G+ extravillous trophoblast cells, which vigorously invade in three-dimensional cultures. Analysis of the methylome reveals that the organoids closely resemble normal first trimester placentas. This organoid model will be transformative for studying human placental development and for investigating trophoblast interactions with the local and systemic maternal environment.Centre for Trophoblast Reearch Royal Society Dorothy Hodgkin Fellowship Marie Curie Intra-European Fellowshi

Research data supporting "Generation of a three-dimensional collagen scaffold-based model of the human endometrium"

The Excel file contains the following information on separate sheets: (1) the volume percent of pore sizes for each scaffold condition, (2) average scaffold pore sizes and standard deviations, and (3) scaffold percolation diameters. These Micro-CT data were collected using the three-dimensional analysis function and the shrink wrap feature on CTAn software, included in the Skyscan package. For percolation diameter calculations, all values were non-negative with R2 > 0.8. The Excel file also includes the decidual stromal cell densities within (4) the top 100 m of the scaffold surfaces for each scaffold condition and (5) a cross-section of the scaffold perpendicular to the scaffold surfaces. The number of cells on the scaffold surfaces was determined by nuclear stains counted by Fiji’s automatic particle analyzer and the TrackMate plugin. Four images parallel to the scaffold surfaces with dimensions of 1.417 mm x 1.417 mm and a thickness of 100 m from the scaffold surface were captured for each scaffold and averaged together. The location of cells within the scaffolds was determined from the coordinates of nuclear stains using the Fiji plugin TrackMate for confocal images taken perpendicularly to the scaffold surfaces. Sheet (6) of the Excel file contains the raw measurements for the sizes of the organoid fragments immediately after fragmentation, as measured on Fiji using a light microscope image. See the main manuscript for more details.This work was supported by the Centre for Trophoblast Research and the Wellcome Trust (090108/Z/09/Z, 085992/Z/08/Z); Y.A was supported by an Isaac Newton grant awarded to M.Y.T; L.G.B. was funded by a Marshall Scholarship from the Marshall Aid Commemoration Commission; M.Y.T. is supported by a Royal Society Dorothy Hodgkin Fellowship; S.M.B. and R.E.C. acknowledge funding from EPSRC Established Career Fellowship Grant No. EP/N019938/1

Human uterine natural killer cells regulate differentiation of extravillous trophoblast early in pregnancy

In humans, balanced invasion of trophoblast cells into the uterine mucosa, the decidua, is critical for successful pregnancy. Evidence suggests that this process is regulated by uterine natural killer (uNK) cells but how they influence reproductive outcomes is unclear. Here, we used our trophoblast organoids and primary tissue samples to determine how uNK cells affect placentation. By locating potential interaction axes between trophoblast and uNK cells using single-cell transcriptomics, and in vitro modelling of these interactions in organoids, we identify a uNK cell-derived cytokine signal that promotes trophoblast differentiation at the late stage of the invasive pathway. Moreover, it affects transcriptional programs involved in regulating blood flow, nutrients, and inflammatory and adaptive immune responses, as well as gene signatures associated with disorders of pregnancy such as pre-eclampsia. Our findings suggest mechanisms on how optimal immunological interactions between uNK cells and trophoblast enhance reproductive success.EU Horizon 202

Mapping the temporal and spatial dynamics of the human endometrium in vivo and in vitro.

The endometrium, the mucosal lining of the uterus, undergoes dynamic changes throughout the menstrual cycle in response to ovarian hormones. We have generated dense single-cell and spatial reference maps of the human uterus and three-dimensional endometrial organoid cultures. We dissect the signaling pathways that determine cell fate of the epithelial lineages in the lumenal and glandular microenvironments. Our benchmark of the endometrial organoids reveals the pathways and cell states regulating differentiation of the secretory and ciliated lineages both in vivo and in vitro. In vitro downregulation of WNT or NOTCH pathways increases the differentiation efficiency along the secretory and ciliated lineages, respectively. We utilize our cellular maps to deconvolute bulk data from endometrial cancers and endometriotic lesions, illuminating the cell types dominating in each of these disorders. These mechanistic insights provide a platform for future development of treatments for common conditions including endometriosis and endometrial carcinoma