2.1 Isolation of hAEC

Ten human term placentas were collected from healthy women undergoing caesarean section after obtaining informed written consent according to the guidelines set by the Ethics Committee of the University of Chieti–Pescara. Placentas were dissected, and hAM was peeled off from four different concentric regions, identified regions according to their macroscopic characteristics and position relative to umbilical cord (Figure S1A): R1 = surrounding the umbilical cord; R2 = intermediate between R1 and R3; R3 = peripheral to the placental disc; R4 = reflected hAM.10 Human amniotic epithelial cells were isolated via trypsin/EDTA 0.05% digestion as previously described.11 Cell viability was assessed by Trypan Blue dye exclusion and consistently exceeded 90%.

2.2 Hepatic differentiation of hAEC

After isolation, hAEC were maintained for 3-6 days in Dulbecco's Modified Eagle Medium (DMEM high glucose, Thermo Fisher Scientific) with standard supplements (Std) defined as follows: 10% FBS, 1% L-glutamine (200 mmol/L), 1% non-essential amino acids, 55 μmol/L 2-mercaptoethanol, 1% sodium pyruvate (100 mmol/L) and 1% antibiotic/antimycotic (all from Thermo Fisher Scientific) and further supplemented with 10 ng/mL epidermal growth factor (Peprotech).

Hepatic differentiation was induced as previously described4 with some modifications (Figure S1B). Culture plates were coated with three different substrates: laminin 521, 422 (both from BioLamina) or Geltrex (Thermo Fisher Scientific). All substrates were used at a final concentration of 10 μg/mL and were allowed to polymerize (overnight at 4°C for laminins, 30 minutes at 37°C for Geltrex). Sub-confluent hAEC monolayers were treated with 0.25% Trypsyn/EDTA solution, and isolated cells were seeded on previously coated plates at a density of ~1 × 10⁵ cells/cm² in DMEM Std + 10 ng/mL EGF and cultured for 24 hours. Cells were then overlayed with 0.44 mg/mL of Geltrex and cultured for 48 hours. Medium was switched to IMDM Std + 10% FBS + 10 ng/mL EGF + 10 ng/mL bFGF (Peprotech) and cultured for 2 days, then added with 20 ng/mL HGF (Peprotech), 1 μmol/L Dex (Sigma Aldrich) 1× Insulin/Transferrin/Selenium (ITS) (Thermo Fisher Scientific) and cultured for further 5 days. For an additional week, the treatment was maintained with the exception of FGF-2 which was replaced by 20 ng/mL oncostatin-M (OSM) (Peprotech).

2.3 Additional materials and methods

Detailed description of materials and methods for transmission electron microscopy, RNA isolation, qRT-PCR, immunofluorescence and statistical analysis is provided in supporting information.

3.1 Laminin 521 enhances hepatic differentiation of hAEC

Previous studies on the differentiation of hAEC into hepatocytes utilized in-house manufactured pig liver-derived extracellular matrix (ECM) as a culture substrate.4 In this study, we sought to identify a suitable commercially available alternative substrate. Engelbreth-Holm-Swarm tumour-derived ECM (commercially known as Matrigel or Geltrex) has long been described as a suitable substrate for the maintenance of primary human hepatocyte cultures.12 More recently, recombinant laminins, major components of basal membranes, have been shown to successfully maintain hepatic function in human hepatocytes in vitro.13 Moreover, laminin 411 and 521 were used for the differentiation and maintenance of induced pluripotent stem cells (iPS)-derived hepatocytes.14, 15 We tested the efficacy of laminin 411, 521 and Geltrex as a culture substrate for the differentiation of hAEC into hepatocyte-like cells. While the morphology was more similar to mature hepatocytes when cells were cultured on Geltrex (Figure 1A-C), gene expression analysis revealed that hAEC-derived hepatocyte-like cells (hAEC-Hep) had higher levels of hepatic marker genes (ie albumin, UDP glucuronosil transferase [UGT1A1]) when cultured on laminin 521 as compared to laminin 411 or Geltrex (Figure 1D).

3.2 hAEC from R1 and R4 are more prone to hepatic differentiation

In order to evaluate the hepatic differentiation ability of hAEC isolated from the four regions, cells were cultured on laminin 521 in the presence of inducive culture conditions for 2 weeks.

Phase contrast analysis of cell morphology showed significant differences between undifferentiated hAEC and hAEC-Hep, although no apparent differences could be observed among cells from different regions (Figure S2). Ultrastructural analysis (Figure 1E-H) of hAEC-Hep monolayers showed the presence of round/polygonal epithelial cells with round central euchromatic nuclei with one or more evident nucleoli in all the 4 regions of the amniotic membrane. The cell cytoplasm of hAEC-Hep displayed morphological characteristics commonly found in hepatocytes in all 4 regions, but mainly in R1 and R3. Such features included an evident rough endoplasmic reticulum and a number of vacuoles and dilated cisternae of smooth endoplasmic reticulum. Interestingly, electron microscopy revealed the presence of glycogen granules (dark rosettes) exclusively in the cytoplasm of hAEC-Hep from R1 inside the dilated cisternae of smooth endoplasmic reticulum that are known to be involved in glycogen metabolism (Figure 1E, inset).

Gene expression analysis revealed that hAEC-Hep derived from R4 had significantly higher levels of albumin, and higher levels of hepatocyte nuclear factor 4 alpha (HNF4α), although this was not statistically significant (Figure 2A). On the other hand, cells from R1 expressed significantly higher levels of cytochrome P450 (CYP) 3A7 and 3A4 and higher levels of alpha-1-antitrypsin (A1AT) as compared to other regions (Figure 2A). A detailed comparison with gene expression levels of foetal and adult human hepatocytes is available in Table S2.

Further investigation with immunofluorescence confirmed that hAEC-Hep from R4 had higher protein expression of albumin and HNF4α, whereas higher levels of CYP 4A were detected in cells from R1 (Figure 2B and Figure S3). Moreover, specific sub-membrane localization of β-catenin was prominent in hAEC-Hep from R4 and, at lower levels, in those from R1 (Figure 2B).