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.

PURPOSE.To compare the effect of epidermal growth factor (EGF), nerve growth factor (NGF), platelet-derived growth factor-BB (PDGF-BB), bovine pituitary extract, and fetal bovine serum (FBS), alone or in combination, on proliferation of human corneal endothelial cells (HCEC) cultured from young (Ͻ30 years old) and older donors (Ͼ50 years old). METHODS. Corneas from donors 2 to 79 years old were obtained from the National Disease Research Interchange. Descemet's membrane with intact endothelium was dissected. Cells were isolated by EDTA treatment and cultured to confluence. The HCEC marker, antibody 9.3.E, tested for pure endothelial populations. Antibody Ki67 and ZO-1 tested either before or after cultured cells reached confluence to indicate cell proliferation and cell-cell contact formation. Cell morphology was documented by inverted phase-contrast microscopy. Passages I through VII were used to test the effect of various factors on cell proliferation. For each study, equal numbers of cells were seeded, maintained overnight in 4% FBS to permit cell attachment, washed, and incubated for up to 3 weeks in one of the following: modified Eagle's Minimum Essential Medium (Opti-MEM-I) alone; Opti-MEM-I plus EGF, NGF, PDGF-BB, bovine pituitary extract, or FBS; or a combination of factors. At various times after seeding, cell numbers were determined by electronic cell counter. For each condition, three separate wells were tested and each sample was counted three times. Studies were repeated at least twice using cells from different donors and age groups. Within each study, a one-way ANOVA test was performed to analyze statistical significance. RESULTS. Cells stained positively with antibody 9.3.E, indicating isolation of HCEC and lack of contamination with epithelial cells or keratocytes. Positive staining of Ki67, indicating cycling cells, was found in subconfluent cultures. Plasma membrane-associated ZO-1 staining and lack of Ki67 staining indicated that cultured cells formed a contact-inhibited monolayer. Cultured cells decreased in density, increased in size, and became more heterogeneous depending on donor age and on the number of passages. Incubation in OptiMEM-I promoted attachment and induced a moderate proliferative response above that of MEM (P Ͻ 0.001). In general, proliferative responses to growth stimuli were relatively slow, with cell counts generally plateauing 10 to 14 days after exposure to growth-promoting agents. EGF yielded a broad, dose-dependent effect and, at 5-50 ng/mL, peak cell counts were significantly higher (P Ͻ 0.001) than basal levels. EGF consistently stimulated proliferation in cells from younger donors, but was less effective in stimulating growth of cells from older donors. NGF did not show a consistent significant stimulatory effect at any concentration tested. PDGF-BB (25 ng/mL) tended to stimulate growth to a greater extent than EGF (P Ͻ 0.05) in cultures from the same donor. Pituitary extract significantly increased counts at 1.0 (P Ͻ 0.05) to 100 ug/mL (P Ͻ 0.001). PDGF-BB plus p...