Step-by-step protocol for isolating the entire repertoire of human first trimester placental cells.

Correct placental development and function are essential for adapting the mother to the ongoing pregnancy and the wellbeing of the growing fetus; however, underlying processes are still poorly understood. Only limited structural and cellular placental features are shared among species hence requiring reliable human in-vitro models. Recently established trophoblast stem cell and organoid models significantly improved placental research; however, the human placenta constitutes a multi-cellular organ with tightly orchestrated, cellular and molecular networks between trophoblasts (TBs) and villous core cells (VCCs) vital for correct placentation. The establishment of co-culture models is accordingly the logical consequence to investigate TB and VCC interactions, but first requires efficient purification of ideally donor-matched placental cell types. We herein present a meticulously-tailored protocol based on four sequential digestion steps (d-steps) with varying enzyme compositions and digestion mode and length, gently releasing cells layer-by-layer from human first trimester placentae (8 - 9th week of gestation). Using immunofluorescence and flow cytometry, we analyzed the tissue fragments and digestion solutions after every d-step and collected data on individual digestion progress as well as cell viability, counts, and specifications. D-step 1 revealed a significantly low viability and was mainly composed of syncytial fragments, extravillous trophoblasts EVTs, and maternal leukocytes. D-step 2 and 3, comprising high viability predominantly contained TBs (90-99 %) with a significant enrichment of EVTs in d-step 2 and an almost pure villous cytotrophoblast (vCTB) population in d-step 3. D-step 4 finally enabled isolating fetal VCCs consisting of endothelial cells, fibroblasts, and Hofbauer cells. Interestingly, maternal leukocytes were detected in d-step 1 and 2 but completely absent from d-step 3 and 4 revealing pure fetal cell populations. In sum, we present a detailed guideline for stepwise isolating selected placental cell types suitable for further studies and co-culture models investigating TB and VCC interactions involved in early placental development.

Generation of extracellular fluids from first-trimester decidual tissues and their validation by detecting tissue-specific secreted proteins.

The human placenta comes in direct contact with maternal cells and blood at two interfaces. The syncytiotrophoblast layer is surrounded by maternal blood at the intervillous space, and extravillous trophoblasts breach the vascular endothelial cells layer upon spiral artery remodeling and invasion of decidual veins. However, little knowledge exists about EVT-derived secreted factors, which may serve as predictive markers for obstetrical syndromes or shape the local environment at the maternal-fetal interface. Here, we define secreted EVT-associated genes and describe a method that yields interstitial fluids from patient-matched first-trimester decidua basalis and parietalis tissues.

The human placenta shapes the phenotype of decidual macrophages.

During human pregnancy, placenta-derived extravillous trophoblasts (EVTs) invade the decidua and communicate with maternal immune cells. The decidua distinguishes into basalis (decB) and parietalis (decP). The latter remains unaffected by EVT invasion. By defining a specific gating strategy, we report the accumulation of macrophages in decB. We describe a decidua basalis-associated macrophage (decBAM) population with a differential transcriptome and secretome compared with decidua parietalis-associated macrophages (decPAMs). decBAMs are CD11chi and efficient inducers of Tregs, proliferate in situ, and secrete high levels of CXCL1, CXCL5, M-CSF, and IL-10. In contrast, decPAMs exert a dendritic cell-like, motile phenotype characterized by induced expression of HLA class II molecules, enhanced phagocytosis, and the ability to activate T cells. Strikingly, EVT-conditioned media convert decPAMs into a decBAM phenotype. These findings assign distinct macrophage phenotypes to decidual areas depending on placentation and further highlight a critical role for EVTs in the induction of decB-associated macrophage polarization.