Journal: Molecular Human Reproduction

Article Title: ADAM8 localizes to extravillous trophoblasts within the maternal–fetal interface and potentiates trophoblast cell line migration through a β1 integrin-mediated mechanism

doi: 10.1093/molehr/gay034

Figure Lengend Snippet: ADAM8 preferentially localizes to anchoring columns of placental villi. (A) Representative immunofluorescence, immunohistochemistry and RNAScope microscopy images of serially sectioned first trimester placental villi (6–11 weeks gestation; n = 5) and decidua (10–12 weeks gestation; n = 4) showing ADAM8 mRNA transcript localization (pink punctate signal) within trophoblast subtypes. Proximal and distal column, as well as interstitial EVT subtypes of trophoblasts are identified by immunostaining of cytokeratin (KRT7) and HLA-G. ‘PCT’ indicates proximal column trophoblast; ‘DCT’ indicates distal column trophoblast; ‘VT’ indicates villous trophoblast; ‘SynT’ indicates syncytiotrophoblast; ‘MC’ indicates placenta mesenchymal core; ‘iEVT’ indicates interstitial extravillous trophoblast; ‘GC’ indicates trophoblast giant cell. The perforated white box indicates enlarged inset image. Black arrowheads denote ADAM8 signal. Bars = 100 μm. (B) Fluorescence activated cell-sorting (FACS) plots demonstrate the trophoblast isolation strategy used to purify mesencymal core cells (MC), distal column trophoblasts (DCT) and villous/proximal column trophoblasts (VT). Live cells, depleted of CD31+ (endothelial) and CD45+ (immune) cells using immuno-magnetic beads, were positively gated by 7AAD exclusion. Cells were further segregated by excluding CD45+ immune cells and by cell surface labeling of HLA-G and CD49f. Cell subtype proportions are indicated within each gated population (percent of cells within FACS plot). (C) ADAM8 mRNA levels in FACS-purified MCs, VTs and DCTs. Trophoblast subtype purity was assessed by qPCR analysis targeting the pan-trophoblast marker KRT7, the EVT-marker HLA-G and the mesenchymal lineage marker VIM. GAPDH was used for normalization. Results are presented as mean ± SD in bar graphs from four distinct placental villi specimens (n = 4); results were analyzed by one-way ANOVA and Dunn’s multiple comparisons test. (D) Immunoblot showing protein levels of pro- and active-ADAM8, HLA-G, and EGFR in MC, VT and DCT isolated from placental villi using immuno-magnetic beads. Molecular weights (kDa) are shown to the left and β-actin indicates loading control.

Article Snippet: Contaminating immune cells were removed from the cell admixture by EasySep CD45 immuno-magnetic bead purification (StemCell Technologies) where remaining cells were plated onto plastic culture plates for 30 min to remove stromal cells.

Techniques: Immunofluorescence, Immunohistochemistry, RNAscope, Microscopy, Immunostaining, Fluorescence, FACS, Isolation, Magnetic Beads, Labeling, Purification, Marker, Western Blot, Control