Differential response to hepatic differentiation stimuli of Amniotic Epithelial Cells isolated from four regions of the Amniotic Membrane

Human Amniotic Epithelial Cells (hAEC) isolated from term placenta are a promising source for regenerative medicine. hAEC can be differentiated into various cell types, including hepatocytes1. However, it has long been debated whether the hAEC population consists of heterogeneous or homogeneous cells. In a previous study, we investigated the characteristics of hAEC isolated from four different regions of the amniotic membrane (AM). We observed morphological and ultrastructural heterogeneity, together with differences in the expression of pluripotency markers and the early hepatic marker ?-fetoprotein. These results suggest that hAEC isolated from different regions of AM could be more or less prone to differentiation towards a specific cell type. The aim of this study was to evaluate the hepatic differentiation of hAEC isolated from these four regions. Term placentae from healthy (n=4) and GD (gestational diabetes) (n=3) women were collected after caesarean section. hAEC were isolated through 0.05% Trypsin digestion from four different regions: R1 (closest to the umbilical cord); R2 (intermediate); R3 (peripheral to the placental disc); R4 (reflected amnion). hAEC were seeded on laminin 411, 521 or Geltrex and treated in hepatic differentiation conditions for 14 days1. hAEC-derived hepatocyte-like cells (hAEC-Hep) differentiated on Geltrex showed a better hepatocyte morphology. However, qRT-PCR showed that hAEC-Hep cultured on laminin 521 had higher expression of hepatic marker genes. When hAEC-Hep subpopulations derived from healthy patients were differentiated on laminin 521, marked differences in gene expression were observed: R4 showed significantly higher levels of Albumin, and high levels of Hepatocyte Nuclear Factor (HNF) 4? and UDP glucuronosyl transferase (UGT) 1A1, whereas R1 expressed significantly higher Cytochrome P450 (CYP) 3A7 and 3A4, and high levels of Alpha-1-Antitrypsin. On the other hand, hAEC-Hep derived from GD patients showed minimal and inconsistent differences. Consistently with qRT- PCR, immunofluorescence analysis of hAEC-Hep from healthy patients revealed that higher levels of Albumin and HNF4? were expressed in R4, whereas higher levels of CYP 4A were detected in R1. Membrane expression of ?-catenin was detected in R4 and, at lower levels, in R1. These differences in protein expression were not detected in hAEC-Hep from GD patients. Moreover, we observed a consistent increase, during differentiation, in Albumin release only in R4, as assessed with ELISA. No differences were observed for hAEC-Hep from GD patients. These preliminary data suggest that healthy patient-derived hAEC isolated from R1 and R4 of the AM are more prone to hepatic differentiation. Furthermore, the pathological state of the patient (i.e. gestational diabetes) could alter these differences. Therefore, the use of hAEC from a specific region of AM should be taken into consideration as it could have an impact on the outcome of therapeutic applications.