Enhanced hepatic differentiation in the subpopulation of human amniotic stem cells under 3D multicellular microenvironment

Figure 1 Characteristics of human amniotic membrane and amniotic epithelial cells. A: H and E staining of amniotic membrane. Bar, 50 µm; B: AP staining of amniotic membrane. Positive cells are indicated with arrows. Bar, 100 µm. In A and B, the amniotic membrane was rolled before embedding. Therefore, many layers can be seen in one picture; C: Immunofluorescent staining of frozen section of amniotic membrane. Anti-SSEA4 antibody (green), E-cadherin antibody (red), and DAPI were used; D: Same as C. Anti-TRA-1-81 antibody (green), anti-EPCAM antibody (red), and DAPI were used; E: Direct tissue staining of amniotic membrane. Anti-TRA-1-60 antibody (green) and DAPI were used. Bars in C, D, and E represent 100 µm; F: Colonies formed from cultured amniotic epithelial cells (AECs) and observed by phase-contrast microscopy. Bar to left of F represents 200 µm. Bar to right of F represents 500 µm; G: Frequency of colony formation from primary AECs and adherent AECs. Cells which did not attach to the well surface were removed to purify the amniotic stem cells; H: Gene expression of primary AECs, mesenchymal stem cells(MSCs), and induced pluripotent stem cells(iPSCs) detected by qRT-PCR; I: surface markers of primary AECs verified by flow cytometry.

Figure 2 Characteristics of primary amniotic epithelial cells and other cell sources verified by bioinformatics. A: Principal component analysis of all cell sources used in this assay; B: General heatmap for each cell source; C: Specific heatmap of stemness and hepatic markers. AEC: Amniotic epithelial cell; Hepa: Hepatocyte; hiHep: Human fibroblast-derived hepatocyte-like cell; MSC: Mesenchymal stem cell; DE: Definitive endoderm; hESC: Human embryonic stem cell; iPSCHLC, iPSC-derived hepatocyte-like cell; NiPS: Normal human iPSC.

Figure 3 Characteristics of amniotic epithelial cell spheres formed on 3D-micropattern plate. A: 3D-micropattern plate used in the present study. Round pits 500 µm in diameter are clustered on the surface. After culture, the amniotic epithelial cells (AECs) formed a sphere; B: Gene expression in the AEC sphere verified by qRT-PCR; C: After reseeding AEC sphere onto 2D culture dish, AEC proliferation was verified by phase-contrast microscopy. Bar, 500 µm.

Figure 4 Organoid morphology and hepatic function. A and C: Frozen sections of amniotic epithelial cell (AEC) sphere and organoid. The AEC sphere was placed in the upper layer and the organoid was placed in the lower layer. H&E staining is used in A, and Periodic acid Schiff staining is used in C; B: Immunofluorescent organoid staining observed under confocal microscopy. Anti-SSEA4 antibody (green) representing AECs; anti-CD90 antibody (red) representing mesenchymal stem cells and DAPI. Bars in A, B, and C, 50 µm; D: ICG tests on AEC sphere and organoid. The AEC sphere was placed in the upper layer and the organoid was placed in the lower layer.