The placenta is crucial for a successful pregnancy and the health of both the fetus and the pregnant woman. However, how the human trophoblast lineage is regulated, including the categorization of the placental cell subtypes is poorly understood. Here we performed single-cell RNA sequencing (RNA-seq) on sorted placental cells from first- and second-trimester human placentas. New subtypes of cells of the known cytotrophoblast cells (CTBs), extravillous trophoblast cells (EVTs), Hofbauer cells, and mesenchymal stromal cells were identified and cell-type-specific gene signatures were defined. Functionally, this study revealed many previously unknown functions of the human placenta. Notably, 102 polypeptide hormone genes were found to be expressed by various subtypes of placental cells, which suggests a complex and significant role of these hormones in regulating fetal growth and adaptations of maternal physiology to pregnancy. These results document human placental trophoblast differentiation at single-cell resolution and thus advance our understanding of human placentation during the early stage of pregnancy.

Proper differentiation of trophoblast cells in the human placenta is a prerequisite for a successful pregnancy, and dysregulation of this process may lead to malignant pregnancy outcomes, such as preeclampsia. Finding specific markers for different types of trophoblast cells is essential for understanding trophoblast differentiation. Here, we report that placenta-specific protein 8 (PLAC8) is specifically expressed in the interstitial extravillous trophoblast cells (iEVTs) on the fetomaternal interface. Using model systems, including placental villi-decidua co-culture, iEVTs induction by using primary trophoblast cells or explants, etc., we found that PLAC8 promotes invasion and migration of iEVTs. Mechanistically, time-lapse imaging, GTPase activity assay, co-immunoprecipitation and RNA-seq studies show that PLAC8 increases the Cdc42 and Rac1 activities, and further induces the formation of filopodia at the leading edge of the migratory trophoblast cells. More interestingly, PLAC8 is significantly upregulated under hypoxia and expression of PLAC8 is higher in iEVTs from preeclamptic placentas when compared with those from the normal control placentas. Together, PLAC8 is a new marker for iEVTs and plays an important role in promoting trophoblast invasion and migration.

Human trophoblast syncytialization, a process of cell-cell fusion, is one of the most important yet least understood events during placental development. Investigating the fusion process in a placenta in vivo is very challenging given the complexity of this process. Application of primary cultured cytotrophoblast cells isolated from term placentas and BeWo cells derived from human choriocarcinoma formulates a biphasic strategy to achieve the mechanism of trophoblast cell fusion, as the former can spontaneously fuse to form the multinucleated syncytium and the latter is capable of fusing under the treatment of forskolin (FSK). Live-cell imaging is a powerful tool that is widely used to investigate many physiological or pathological processes in various animal models or humans; however, to our knowledge, the mechanism of trophoblast cell fusion has not been reported using a live-cell imaging manner. In this study, a live-cell imaging system was used to delineate the fusion process of primary term cytotrophoblast cells and BeWo cells. By using live staining with Hoechst 33342 or cytoplasmic dyes or by stably transfecting enhanced green fluorescent protein (EGFP) and DsRed2-Nuc reporter plasmids, we observed finger-like protrusions on the cell membranes of fusion partners before fusion and the exchange of cytoplasmic contents during fusion. In summary, this study provides the first video recording of the process of trophoblast syncytialization. Furthermore, the various live-cell imaging systems used in this study will help to yield molecular insights into the syncytialization process during placental development.

BACKGROUND The literature on post-hepatectomy bile duct injury (PHBDI) is limited, lacking large sample retrospective studies and high-quality experience summaries. Therefore, we reported a special case of iatrogenic bile duct injury caused by Glissonean pedicle transection with endovascular gastrointestinal anastomosis (endo-GIA) during a right hepatectomy, analyzed the causes of this injury, and summarized the experience with this patient. CASE SUMMARY We present the case of a 66-year-old woman with recurrent abdominal pain and cholangitis due to intrahepatic cholangiectasis (Caroli's disease). Preoperative evaluation revealed that the lesion and dilated bile ducts were confined to the right liver, with right hepatic atrophy, left hepatic hypertrophy, and hilar translocation. This problem can be resolved by performing a standard right hepatectomy. Although the operation went well, jaundice occurred soon after the operation. Iatrogenic bile duct injury was considered after magnetic resonance cholangiopancreatography review, and the second operation were performed 10 d later. During the second operation, it was found that the endo-GIA had damaged the lateral wall of the hepatic duct and multiple titanium nails remained in the bile duct wall. This led to severe stenosis of the duct wall, and could not be repaired. Therefore, the injured bile duct was transected, and a hepatic-jejunal-lateral Roux-Y anastomosis was performed at the healthy part of the left hepatic duct. After this surgery, the patient had a smooth postoperative recovery, and the total bilirubin gradually decreased to normal. The patient was discharged 41 d after operation. No anastomotic stenosis was found at the 6 mo of follow-up. CONCLUSION Not all cases are suitable for endo-GIA transection of Glissonean pedicle, especially in cases of intrahepatic bile duct lesions. PHBDI caused by endo-GIA is very difficult to repair due to extensive ischemia, which requires special attention.