Mapping the Human Amniotic Membrane: Useful Instructions for the Selection of Cells in Regenerative Medicine Approaches

The placenta is an important organ during pregnancy for the maintenance of feto-maternal tolerance, which is removed and discarded after childbirth. In recent years, there has been a growing interest in stem cells from the placenta, since it is a useful source of cells for the treatment of diseases in regenerative medicine without any ethical problems. In this study, we analyzed the amniotic membrane from the human placenta with the aim to map different regions with respect to the structure properties and regenerative potential. We dissected placentas of 6 healthy women (31.16 ± 6.3) that underwent a cesarean section at the hospital of Chieti as well as at the hospital of Brescia, to obtain samples for light and electron microscopy. In immunohistochemistry, we analyzed the expression of different markers of pluripotency and proliferation (OCT-4, c-KIT, SOX-2, ?-fetoprotein, CREB and p-CREB). Three areas were considered according to their position in relation to the umbilical cord, so that the first area was closer to the umbilical cord (the central area), the second and larger in the middle (the intermediate area), and the third was the most distant (the peripheral area). Morphometric analysis demonstrated that the OCT-4 and CREB expression in amniotic epithelial nuclei and cytoplasm was significantly higher in the peripheral area than in the intermediate area, whereas the central area had the weakest expression. Moreover, both the c-KIT and SOX-2 expression in amniotic epithelial cells, as well as p-CREB expression, were higher in the peripheral area, while ?-fetoprotein expression was significantly higher in the central area. Similar results were obtained in amniotic mesoderm with respect to OCT-4 and ?-fetoprotein expression, whereas no differences were found between the three areas in SOX-2 expression. Interestingly, the amniotic epithelium from both the central area and the peripheral area was occasionally multi-layered and displayed detaching cells or cell cytoplasm fragments, indicating the existence of amniotic membranederived exosomes. In consistence with these observations, electron microscopy analysis displayed a greater presence of lipid granules in epithelial cells from the peripheral area of amniotic membrane. Our morphological and histo-chemical analysis highly suggests that the different areas of amniotic membrane are characterized by different cells types that could present a different regenerative potential. Functional assays are being performed to confirm our preliminary observations with the aim to increase efficiency of amniotic membrane engraftment within a therapeutic context