Fig 1.
Mouse liver decellularisation.
(a) Macroscopic appearance of a mouse liver scaffold during the decellularisation process. Following the perfusion with demineralized water (dH2O), the fresh red liver becomes blanched. After perfusion with sodium deoxycholate (SDC) followed by DNAseI solution, the liver becomes transparent. (b) H&E staining of the liver sections shows no cells in the scaffold after DET decellularisation. (c) DNA quantification shows a significant (*) reduction of DNA amount in the DET liver (median = 0.093 μg/mg) compared to fresh tissue (median = 1.068 μg/mg) (nFresh = 4, nDET = 4; α = 0.05; p = 0.029; two-tailed Mann-Whitney test). (d) Scanning Electron Microscopy demonstrating the preservation of the three-dimensional network of connective tissue fibres.
Fig 2.
Characterisation of the mouse liver scaffold decellularised by DET method.
(a) DAPI (grey) staining of sections of a DET mouse liver scaffold shows efficient decellularisation of the fibres with no nucleus present. (b) Picosirius red (PR) staining shows an abundance of collagen fibres in the scaffold. (c) Alcian Blue (AB) staining demonstrates presence of glycosaminoglycans (GAG). (d) Elastin van Gieson staining (EVG) confirms presence of elastin.
Fig 3.
Differentiation of hiPSCs and hESCs in 2D or 3D cultures.
hiPSCs and hESCs were differentiated first towards the definitive endoderm. Definitive Endoderm-like cells (DECs) were then harvested and either seeded on matrigel-coated petri dishes (2D cultures) or injected into multiple locations in individual decellularised liver lobes (3D cultures) on day 6. The differentiation protocol was continued in parallel in both systems with the hepatic specification stage from d7-d11 followed by the hepatic maturation stage from d11. The differentiation was continued up to day 13 when the samples were analysed. Part of the illustrations were published in Maghsoudlou et al [26].
Fig 4.
Repopulation of a mouse decellularised liver scaffold with hiPSC- and hESC-derived hepatocytes on day 13 of differentiation.
(a-b) Nuclear staining by DAPI shows that cells have attached along the fibres of the scaffold (a) and that they repopulated the hepatic spaces as shown by H&E staining (b). (c) Scanning Electron Microscopy demonstrates engraftment of cells within the hepatic spaces of the scaffold. (d) Ki67 (proliferation marker) staining of sections of mouse liver scaffold repopulated with hESC- or hiPSC-derived hepatocytes. Nuclei counterstained with DAPI.
Fig 5.
Minimal apoptosis amongst hiPSC- and hESC-derived hepatocytes cultured in 3D on day 13 of differentiation.
Cleaved caspase 3 (red) staining demonstrates only occasional apoptotic cells. Nuclei stained with DAPI.
Fig 6.
Morphology of hiPSC- or hESC-derived cells in 2D on day 13 of differentiation.
Phase contrast microscopy shows cells starting to acquire a polygonal shape.
Fig 7.
ALB and AFP expression by hiPSC- or hESC-derived hepatocytes on day 13 of differentiation.
(a-b) Immunostaining of ALB shows more widespread expression in 3D (b) than in 2D (a). Nuclei stained with DAPI. (c-d) Immunostaining shows fewer cells expressing AFP in 3D (d) than in 2D (c). Nuclei stained with DAPI. (e-f) Boxplots of percentages of positive cells for ALB (e) and AFP (f) for each group. The bottom and top of the box are the first and third quartiles, and the band inside the box is the median. The ends of the whiskers represent the minimum and maximum. Individual data points represented by ○ and ▲ are mild and extreme outliers, respectively. Statistical difference level is indicated by ** for p<0.01 according to the results of the Dunn-Bonferroni posthoc tests performed with the Kruskal-Wallis test (α = 0.05).
Fig 8.
HNF4α expression by hiPSC- or hESC-derived hepatocytes on day 13 of differentiation.
(a-b) Immunostaining of the hepatoblast and hepatocyte marker HNF4α shows its expression by 2D (a) and 3D (b) cultured cells. Nuclei stained with DAPI. The bottom high magnification panel corresponds to the delineated regions in lower magnification panel above.
Fig 9.
CK-18 and CK-19 expression by hiPSC- or hESC-derived hepatocytes in 2D and 3D on day 13 of differentiation.
(a-b) Immunostaining of CK-18, typically expressed by hepatoblasts and hepatocytes and CK-19, expressed by hepatoblasts and cholangiocytes. Whilst CK-18 staining is seen in both 2D (a) and 3D (b) cultured cells, CK-19 staining is absent in 3D cultures. Nuclei stained with DAPI. (c-d) Boxplots of percentages of positive cells for CK-18 (c) and CK-19 (d) for each group. The bottom and top of the box are the first and third quartiles, and the band inside the box is the median. The ends of the whiskers represent the minimum and maximum. Individual data points represented by ○ and ▲ are mild and extreme outliers, respectively. Statistical difference level is indicated by ** for p<0.01 according to the results of the Dunn-Bonferroni posthoc tests performed with the Kruskal-Wallis test (α = 0.05).
Fig 10.
CK-7 expression by hiPSC- or hESC-derived hepatocytes on day 13 of differentiation.
(a-b) Immunostaining of CK-7, typically expressed by ductal plate cells and cholangiocytes (CK7, green) demonstrates expression in 2D cultured (a) but not 3D cultured (b) cells. Nuclei stained with DAPI.
Fig 11.
ASGPR expression by hiPSC- or hESC-derived hepatocytes in 2D or 3D on day 13 of differentiation.
(a-b) Immunostaining of hepatocyte sinusoidal membrane protein ASGPR in 2D (a) and 3D (b) cultured cells. Nuclei stained with DAPI. The higher magnification images of merged channels in the bottom panel correspond to the delineated lower magnification images in the panel above. Images of 3D cultured cells demonstrate membrane localisation of ASGPR.
Fig 12.
Lipid storage by hiPSC- or hESC-derived hepatocytes in 2D or in 3D on day 13 of differentiation.
(a-b) Lipids detected by ORO (droplets, red-orange). Nuclei stained with hematoxylin (purple). Substantially higher percentage of cells are ORO positive in 3D (b) than in 2D (a) on day 13. (c) Boxplots of percentages of positive cells for ORO for each group. The bottom and top of the box are the first and third quartiles, and the band inside the box is the median. The ends of the whiskers represent the minimum and maximum. Individual data points represented by ○ and ▲ are mild and extreme outliers, respectively. Statistical difference level is indicated by ** for p<0.01 according to the results of the Dunn-Bonferroni posthoc tests performed with the Kruskal-Wallis test (α = 0.05).
Fig 13.
Glycogen synthesis by hiPSC- or hESC-derived hepatocytes on day 13 of differentiation.
Periodic Acid Schiff staining (PAS, purple-magenta) is used to detect intracellular polysaccharides. A higher proportion of PAS positive cells is detected in 3D cultures (b) than in 2D cultures (a) at day 13 of differentiation. Nuclei were stained with hematoxylin (dark purple).