Mapping of the Human Placenta: Experimental Evidence of Amniotic Epithelial Cell Heterogeneity

Abstract

The human placenta is an important source of stem cells that can be easily collected without ethical concerns since it is usually discarded after childbirth. In this study, we analyzed the amniotic membrane (AM) from the human placenta with the aim of mapping different regions with respect to their morpho-functional features and regenerative potential. AMs were obtained from 24 healthy women, undergoing a caesarean section, and mapped into 4 different regions according to their position in relation to the umbilical cord: the central, intermediate, peripheral, and reflected areas. We carried out a multiparametric analysis focusing our attention on amniotic epithelial cells (AECs). Our results revealed that AECs, isolated from the different areas, are a heterogeneous cell population with different pluripotency and proliferation marker expression (octamer-binding transcription factor 4 [OCT-4], tyrosine-protein kinase KIT [c-KIT], sex determining region Y-box 2 [SOX-2], α-fetoprotein, cyclic AMP response element binding [CREB] protein, and phosphorylated active form of CREB [p-CREB]), proliferative ability, and osteogenic potential. Our investigation discloses interesting findings that could be useful for increasing the efficiency of AM isolation and application for therapeutic purposes.

Keywords: amniotic epithelial cells; amniotic membrane; human term placenta; placenta stem cells.

Figure 1.

(A) The histological structure of the human placenta. Representative 2-D reconstruction of the human placenta, stained with trichrome Mallory solution (original magnification: ×2.5), and schematic representation of the placental structure. AE, amniotic epithelium; AM, amniotic mesenchyme; Ch, chorion; D, decidua. In the right panels, details are shown at a higher magnification (×10 and ×40). (B) The histological structure of the different areas of the human amniotic membrane. The pictures show the amniotic epithelium and the underlying connective tissue of the central, intermediate, peripheral, and reflected areas. Black arrows point at apoptotic cells, arrowheads point at multilayered epithelium and detaching cells, and green arrows point at budding cells. Sections were stained with hematoxylin–eosin solution (original magnification: ×20).

Figure 2.

Immunohistochemistry in light microscopy. Photomicrographs depicting the immunohistochemical reaction for OCT-4, c-KIT, SOX-2, α-fetoprotein, CREB, and p-CREB in the central, intermediate, peripheral, and reflected areas (original magnification: ×40). Black arrows point at human amniotic mesenchymal stromal cells (AMSCs). The insets show the negative control of the reaction for all antibodies (original magnification: ×40).

Figure 3.

Quantification and statistical analysis of immunohistochemical reactions. (A) Light microscopy image representative of the method used for quantitative analysis of the immunohistochemical reactions with MetaMorph analysis software (original magnification: ×40). The selected area for each sample was 3,000 μm². (B) Bar graph representation of the immune labeling for OCT-4, SOX-2, c-KIT, α-fetoprotein, CREB, and p-CREB in the central (C), intermediate (I), peripheral (P), and reflected (R) areas of the amniotic membrane. Values reported in the bar graphs are the means ± standard deviation (SD) of labeled area from at least 3 different experiments.*P < 0.05: OCT-4 central area versus peripheral area, α-fetoprotein intermediate area versus peripheral area, and CREB central area versus peripheral area.

Figure 4.

(A): Colony-forming unit assay of amniotic epithelial cells (AECs) isolated from different regions of the amniotic membrane: 1 × 10⁴ or 2 × 10⁴ AECs were seeded per well. Graphs represent mean and standard error mean (SEM). Statistical analysis was performed using GraphPad v6 by a 1-way analysis of variance followed by the Bonferroni multicomparisons test. (B) Osteogenic differentiation of AECs isolated from different regions of the amniotic membrane. Representative images of osteogenic differentiation of AECs evaluated through calcium deposit staining with Alizarin red and Von Kossa staining methods (original magnification: ×4).