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.

Characterization of primary models of human trophoblast.

Two recently developed models, trophoblast organoids and trophoblast stem cells (TSCs), are useful tools to further the understanding of human placental development. Both differentiate from villous cytotrophoblast (VCT) to either extravillous trophoblast (EVT) or syncytiotrophoblast (SCT). Here, we compare the transcriptomes and miRNA profiles of these models to identify which trophoblast they resemble in vivo. Our findings indicate that TSCs do not readily undergo SCT differentiation and closely resemble cells at the base of the cell columns from where EVT derives. In contrast, organoids are similar to VCT and undergo spontaneous SCT differentiation. A defining feature of human trophoblast is that VCT and SCT are human leukocyte antigen (HLA) null, whereas EVT expresses HLA-C, -G and -E molecules. We find that trophoblast organoids retain these in vivo characteristics. In contrast, TSCs express classical HLA-A and HLA-B molecules, and maintain their expression after EVT differentiation, with upregulation of HLA-G. Furthermore, HLA expression in TSCs differs when grown in 3D rather than in 2D, suggesting that mechanical cues are important. Our results can be used to select the most suitable model for the study of trophoblast development, function and pathology.

Establishment and differentiation of long-term trophoblast organoid cultures from the human placenta.

The human placenta is essential for successful reproduction. There is great variation in the anatomy and development of the placenta in different species, meaning that animal models provide limited information about human placental development and function. Until recently, it has been impossible to isolate trophoblast cells from the human placenta that proliferate in vitro. This has limited our ability to understand pregnancy disorders. Generating an in vitro model that recapitulates the unique features of the human placenta has been challenging. The first in vitro model system of human trophoblast that could be cultured long term and differentiated to syncytiotrophoblast (SCT) and extravillous trophoblast (EVT) was a two-dimensional (2D) culture system of human trophoblast stem cells. Here, we describe a protocol to isolate trophoblast from first-trimester human placentas that can be grown long term in a three-dimensional (3D) organoid culture system. Trophoblast organoids can be established within 2-3 weeks, passaged every 7-10 d, and cultured for over a year. The structural organization of these human trophoblast organoids closely resembles the villous placenta with a layer of cytotrophoblast (VCT) that differentiates into superimposed SCT. Altering the composition of the medium leads to differentiation of the trophoblast organoids into HLA-G+ EVT cells which rapidly migrate and invade through the Matrigel droplet in which they are cultured. Our previous research confirmed that there is similarity between the trophoblast organoids and in vivo placentas in their transcriptomes and ability to produce placental hormones. This organoid culture system provides an experimental model to investigate human placental development and function as well as interactions of trophoblast cells with the local and systemic maternal environment.

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.