Proliferative, Migratory, and Transition Properties Reveal Metastate of Human Amnion Cells

Abstract

Amnion epithelial cell (AEC) shedding causes microfractures in human placental membranes during gestation. However, microfractures are healed to maintain membrane integrity. To better understand the cellular mechanisms of healing and tissue remodeling, scratch assays were performed using primary AECs derived from normal term not in labor membranes. AECs were grown under different conditions: i) normal cultures (control), ii) oxidative stress (OS) induction by cigarette smoke extract (CSE), iii) co-treatment of CSE and antioxidant N-acetyl-l-cysteine, and iv) treatment with amniotic fluid (AF). Cell migration time and distance, changes in intermediate filament (cytokeratin-18 and vimentin) expressions, and cellular senescence were determined. Control AECs in culture exhibited a metastate with the expression of both cytokeratin-18 and vimentin. During healing, AECs proliferated, migrated, and transitioned from epithelial to mesenchymal phenotype with increased vimentin. Wound healing was associated with mesenchymal to epithelial transition (MET). CSE-induced OS and senescence prevented wound healing in which cells sustained mesenchymal state. N-acetyl-l-cysteine reversed CSE's effect to aid wound closure through MET. AF accelerated cellular transitions and healing. Our data suggest that AECs undergo epithelial to mesenchymal transition during proliferation and migration and MET at the injury site to promote healing. AF accelerated whereas OS diminished cellular transitions and healing. OS-inducing pregnancy risk factors may diminish remodeling capacity contributing to membrane dysfunction, leading to preterm birth.

Amnion membrane organ‐on‐chip: an innovative approach to study cellular interactions

“…EMT contributes to sustained inflammation that promotes the labor-related cascade of events. Data from this study indeed confirmed our earlier findings (17) that CSE treatment for 48 h induced a fibroblastoid morphology in AECs and vimentin relocalization to the leading edge (white arrow) of migratory cells and significantly increased vimentin:CK-18 ratio (control: 0.56 6 0.02, CSE: 0.67 6 0.02; P = 0.02) compared with AECs under standard cell culture conditions [vimentin (perinuclear)] and CK-18 (cytoplasmic; Fig. 3F, G).…”

“…Thus, the results shows that AECs must undergo 2 cellular transitions, EMT to migrate and MET to exit, to completely migrate through the type IV collagen-filled microchannels. These transitions are similar to what we have reported in scratch assays that resembled membrane MF healing, in which migrating and healing edges had mesenchymal and epithelial phenotypes, respectively (17).…”

“…Cell staining AEC and AMC immunocytochemical staining for vimentin (3.7 ml/ml; ab92547; Abcam, Cambridge, MA, USA) and cytokeratin-18 (CK-18; 1 ml/ml; ab668; Abcam) were performed after 48 h as previously described by Richardson et al (17). Manufacturer's instructions were used to calculate staining dilutions to ensure uniform staining.…”

“…Increased OS at term has been shown to induce laborassociated changes, such as cellular senescence, matrix metallopeptidase 9 up-regulation, and increased proinflammatory cytokine production in fetal membrane cells, including AECs and AMCs (2,23). CSE, a potent and reliable OS inducer (2,17,23,(31)(32)(33)(34)(35), has been shown to recreate the labor phenotype (OS experienced at term labor in amnion membranes) in vitro and to induce a static state of EMT in AECs (17,33). EMT contributes to sustained inflammation that promotes the labor-related cascade of events.…”

“…AECs and AMCs also provide essential immune and endocrine functions that are critical to the maintenance of pregnancy (14)(15)(16). Membrane growth and remodeling are essential during gestation and involve both cellular transitions and collagenolytic matrix turnover (8,17). Cellular-level changes primarily involve AECs and AMCs that are conventionally considered as purely epithelial and mesenchymal in physiognomies.…”