Single-cell characterization of self-renewing primary trophoblast organoids as modeling of EVT differentiation and interactions with decidual natural killer cells

Abstract

Background Extravillous trophoblast cell (EVT) differentiation and its communication with maternal decidua especially the leading immune cell type natural killer (NK) cell are critical events for placentation. However, appropriate in vitro modelling system and regulatory programs of these two events are still lacking. Recent trophoblast organoid (TO) has advanced the molecular and mechanistic research in placentation. Here, we firstly generated the self-renewing TO from human placental villous and differentiated it into EVTs (EVT-TO) for investigating the differentiation events. We then co-cultured EVT-TO with freshly isolated decidual NKs for further study of cell communication. TO modelling of EVT differentiation as well as EVT interaction with dNK might cast new aspect for placentation research. Results Single-cell RNA sequencing (scRNA-seq) was applied for comprehensive characterization and molecular exploration of TOs modelling of EVT differentiation and interaction with dNKs. Multiple distinct trophoblast states and dNK subpopulations were identified, representing CTB, STB, EVT, dNK1/2/3 and dNKp. Lineage trajectory and Seurat mapping analysis identified the close resemblance of TO and EVT-TO with the human placenta characteristic. Transcription factors regulatory network analysis revealed the cell-type specific essential TFs for controlling EVT differentiation. CellphoneDB analysis predicted the ligand-receptor complexes in dNK-EVT-TO co-cultures, which relate to cytokines, immunomodulation and angiogenesis. EVT was known to affect the immune properties of dNK. Our study found out that on the other way around, dNKs could exert effects on EVT causing expression changes which are functionally important. Conclusion Our study documented a single-cell atlas for TO and its applications on EVT differentiation and communications with dNKs, and thus provide methodology and novel research cues for future study of human placentation.

Single-cell and spatial transcriptomics reveal alterations in trophoblasts at invasion sites and disturbed myometrial immune microenvironment in placenta accreta spectrum disorders

“…The cells of scRNA-seq were clustered using t-distributed stochastic neighbor embedding (t-SNE) (Fig. 1B), and the major cell types were identified with the expression of canonical marker genes [19,35,44,45], including three types of trophoblasts: EVT (HLA-G, PAPPA2) [19,[45][46][47][48], STB (GDF15, TFPI) [46,49], CTB (PAGE4) [46,50]; stromal cells: SMC (ACTA2) [35], fibroblast (DCN) [35], endothelial cell (EC) (VWF) [35], lymphatic endothelial cell (LEC) (TFF3) [35]; immune cells: T cell (CD3D) [19,35], B cell (CD79A) [19,35], NK cell (KLRB1) [51], monocyte (CD14) [35,52], mast cell (TPSB2) [35]; cells in cell cycle (cycling) (TOP2A) [53] and red blood cell (RBC) (HBA2) [54] (Fig. 1C and S2B).…”

Hypoxia and the endometrium: An indispensable role for HIF-1α as therapeutic strategies

“…Decidual natural killer (dNK) cells are the dominant group of immune cells recruited in the decidual tissue. During early pregnancy, dNK cells are located adjacent to invading trophoblasts, regulating embryo implantation and maternal immune response [ 45 ]. In dNK cells, α-ketoglutarate inhibits the egl-9 family hypoxia-inducible factor 1-mediated ubiquitination of HIF-1α, which in turn promotes the transcription of insulin-like growth factor 1 and growth differentiation factor 15, contributing to trophoblast invasion [ 46 ].…”

Review on new approach methods to gain insight into the feto-maternal interface physiology

“…While patient-derived tumor organoids and patient-derived spheroids (tumorspheres) have a place in cancer research and precision medicine strategies, the more functionally advanced organoid can be sourced as a living tissue biobank, provide disease-specific models, and (like some spheroids) be useful in drug screening (15)(16)(17). The spheroid and organoids are now being developed and getting used for several FMi research (18)(19)(20)(21)(22)(23)(24)(25)(26)(27). These approaches and tools will be optimized and available widespread for research use in reproductive biology and medicine.…”