Isolation of AE Cells
1. Human placentae were obtained with the approval of the institutional review board, after uncomplicated elective caesarean deliveries from healthy mothers.
2. Amnion layer was prepared according to the methods (reference) with the following modifications.
3. The amnion layer was mechanically peeled off of the chorion and washed several times with Hanks' balanced salt solution (HBSS) without calcium and magnesium to remove blood.
4. To release AE cells, the amnion membrane was incubated at 37°C with 0.05% trypsin containing 0.53 mM EDTA4Na.
5. The cells from the first 10 minutes of digestion were discarded to exclude debris.
6. The cells from the second and third 30-minute digests were pooled and washed three times with HBSS.
7. Viability of the AE cells was determined by exclusion of trypan blue dye and counted with a hemocytometer.

Cell Culture and Standard Culture Media
1. AE cells were plated on 100-mm-diameter cell culture dishes at a density of 12.7 × 10⁴ cells per cm² in our standard culture media containing 10 ng/ml epidermal growth factor (EGF).
2. EGF was defined in preliminary experiments to induce robust proliferation.
3. Within 24–48 hours, AE cells achieved >80% confluency, and the cells were dissociated by trypsin and plated at a density of 1 × 10⁴ cells per cm² on culture dishes for further differentiation protocols.
4. Standard culture media is Stem cell medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1% nonessential amino acid, 55 μM 2-mercaptoethanol, 1 mM sodium pyruvate, and 1% antibiotic-antimycotic.
5. This standard media may be supplemented with a variety of growth factors as indicated in the text.

Isolation of Attached and Intermediate Layers from AE Cultures
1. AE cells were isolated and plated under our standard culture conditions on 100-mm-diameter culture dishes at a density of 1 × 10⁷ cells per dish.
2. After 5 days in culture, the cells in the supernatant, cells in the intermediate layer, and cells attached to the culture dish were collected as separate fractions.
3. The supernatant fraction was collected from aspirated media and washed with HBSS.
4. The middle- or intermediate-layer cells were released with trypsin under careful microscopic observation.
5. Adherent cells attached to the culture dish were isolated by more extended trypsinization.

Fluorescence-Activated Cell Sorter Analysis
Freshly isolated AE cells were examined for surface antigens commonly found on embryonic stem cells (ESCs). The following specific primary monoclonal antibodies (2 μg/ml each) were used to detect surface-antigen expression: SSEA-1 (MAB4301), SSEA-3 (MAB4303), SSEA-4 (MAB4304), TRA1-60 (MAB4360), TRA 1-81 (MAB4381), TRA 2-54 (MAB4354), Thy1.1, c-kit, and CD34.

Reference
1. Akle CA, Adinolfi M, Welsh KI et al. Immunogenicity of human amniotic epithelial cells after transplantation into volunteers. Lancet 1981; 2: 1003–1005.
2. Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998; 282: 1145–1147.
3. Reubinoff BE, Pera MF, Fong CY et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000; 18: 399–404.
4. Toshio Miki, Thomas Lehmann, Hongbo Cai, Donna B. Stolz, Stephen C. Strom. Stem cell characteristics of amniotic epithelial cells. Stem cell. 2005; 23: 1549–1559.