The term basal plate of the human placenta as a source of functional extravillous trophoblast cells

Abstract

Background: Extravillous trophoblast (EVT) cells are of pivotal importance in human embryo implantation and homeostasis of the maternal fetal interface. Invasion of the endometrium by EVT contributes to placental anchorage, spiral artery remodeling, immunological defense, tolerogenic responses, and several collaborative cross talks involved in establishing and maintaining a successful pregnancy. We report here an improved protocol for the isolation of fully differentiated EVT cells from the basal plate of the human term placenta.

Methods: The basal plate was carefully dissected from the villous tissue and the amniochorion membrane prior to enzymatic digestion. Term basal EVT cells were isolated using a 30 and 60% Percoll gradient. A panel of markers and characteristics of the isolated cells were used to confirm the specificity and efficiency of the method so that their potential as an investigative tool for placental research could be ascertained.

Results: Isolated cells were immunoreactive for cytokeratin-7 (CK-7), placental growth factor, placental alkaline phosphatase, human leukocyte antigen G1 (HLA-G1), and α1 and α5 integrins, similarly to the EVT markers from first trimester placental villi. Around 95% of the isolated cells labeled positively for CK-7 and 82% for HLA-G1. No significant change in viability was observed during 48 h of EVT culture as indicated by propidium iodide incorporation and trypan blue test exclusion. Genes for metalloproteinases MMP-2 and MMP9 (positive regulators of trophoblast invasiveness) were expressed up to 48 h of culturing, as also the gelatinolytic activity of the isolated cells. Transforming growth factor (TGF)-beta, which inhibits proliferation, migration, and invasiveness of first-trimester EVT cells, also reduced invasion of isolated term EVT cells in transwell assays, whereas epidermal growth factor was a positive modulator.

Conclusions: Term basal plate may be a viable source of functional EVT cells that is an alternative to villous explant-derived EVT cells and cell lines. Isolated term EVT cells may be particularly useful in investigation of the role of trophoblast cells in pathological gestations, in which the precise regulation and interactive ability of extravillous trophoblast has been impaired.

Figure 1

The term decidual basal plate. (A) Panoramic view (25x) showing the basal plate (BP), chorionic villi (ChV) and the chorionic plate (ChP) of the human placenta. Dotted line indicates the area initially dissected for isolation of EVT cells. (B) High magnification (100x) of the square highlighted in Figure A, in which are indicated extravillous cytotrophoblast cells (EVT), decidual cells (DC) and leukocytes (LK) at the basal plate (BP). (fb) fibrinoid. (ChV) chorionic villi. Hematoxylin-eosin staining.

Figure 2

General morphology of isolated and cultured term basal plate cells. (A) After isolation (200x). (B) Cells organized in cell columns after 24 h of culture on Matrigel (200x). (C) Higher magnification of B (400x). (D-F) After 48 h of culture, cells exhibit projections indicating migratory activity (1000x). (A) Phase-contrast. (B-F) Mayer’s hematoxylin staining.

Figure 3

Representative immunofluorescence characterization and purity of isolated term basal plate cells cultured for 48 h. (A, D) Negative controls of immunostaining. (B) Vimentin staining (200x); insert shows vimentin-expressing endometrial stromal cells (200x). (C) CK-7 staining (200x); insert shows a CK-7 positive cell exhibiting projections (1000x). (E) PlGF staining (400x). (F) PlAP staining (200x). (G) CD68 staining (200x). (H) Alpha6-integrin staining (200x). (I) Desmoplakin I/II staining (200x).

Figure 4

Representative flow cytometry characterization and purity of isolated term basal plate cells cultured for 48 h. Flow cytometry plots show cell size and cytoplasmic granularity (A), the amount of CK-7 positive cells (B), the amount of vimentin positive cells (C) and HLA-G1 positive cells (D). The data are representative of three independent assays.

Figure 5

Term basal plate EVT cells express the main EVT markers similarly to first trimester EVT cells cultured on fibronectin for 48 h. (A,D,G) Negative controls of the immunofluorescence reactions (200x). (B-C) HLA-G staining (200x). (E-F) Alpha1-integrin staining (200x). (H-I) Alpha5-integrin staining (200x).

Figure 6

Cell viability and mRNA expression of term basal plate EVT cells at different times of culture. Cell viability was analyzed by PI incorporation and trypan blue exclusion using complete DMEM/F12 (A) and supplemented IMDM (B). The data represent the mean±SEM of four independent assays. (C) Agarose gel electrophoresis of amplified PCR products of GAPDH, 18S, MMP-2 and MMP-9 genes. (D) Representation of the RT-PCR data as determined by densitometric analysis of gel bands expressed as a ratio of GAPDH expression. The data represent the means±SEM from three independent experiments. **p<0.01; ***p<0.001 versus control.

Figure 7

Term EVT cells maintain their capacity to invade. TGF-β and EGF modulate the invasion of basal plate EVT maintained in (A) Matrigel or (B) fibronectin coated transwell inserts and cultured for 48 h. *p<0.05; **p<0.01; ***p<0.001 versus control. (C) MMP-2 and MMP-9 activities in the presence of TGF-β and EGF in basal plate EVT cultured for 48 h on Matrigel. (D) Results of densitometric analysis of gel electrophoresis expressed as fold change in relation to control cultures. In panels A, B and D the data represent the means±SEM of three independent experiments.