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. Author manuscript; available in PMC: 2020 Dec 14.
Published in final edited form as: Cell Rep. 2020 Jun 2;31(9):107711. doi: 10.1016/j.celrep.2020.107711

Figure 5. Hepatic Function and Characterization of Engineered Human iPSC-Derived Liver Graft.

Figure 5.

(A) Photograph illustrating the organ perfusion and culture chamber used to recellularized rat livers with human iPSC-Heps, iPSC-VECs, human fibroblasts, mesenchymal stem cells, and iPSC-Chol.

(B) Decellularized whole liver matrix (left) and liver after recellularization (right).

(C) Double-immunofluorescence staining for Ki67 and ALB, CD31, and cytokeratin 7 (CK7) of regenerated liver grafts four days after recellularization. Human fetal liver (gestational age; week 16) and adult liver tissues were used as the controls. Bar graphs showing the levels of Ki67 positive cell percentage are also indicated for each cell type.

(D) Immunofluorescence staining for the key markers of cell adhesions and tight junctions, integrin beta-1 (ITGB1), ZO-1, and Conexin32 (CX32), of the regenerated human liver grafts. Bioengineered livers with primary rat liver cells, human fetal liver, human adult liver and rat adult liver were used as controls.

(E) Left: the comparison of bile-acid production between assembled liver grafts derived from rat hepatocytes and assembled liver grafts derived from human iPSCs. Right: regenerated liver tissue assembled with human iPSC-human derived cells (assembled human iPSC-liver) in comparison to that from iPSC-Heps cultured alone in static sandwich (iPSC-Heps [3D]) and human iPSC-Heps cultured with iPSC-VECs, iPS-derived cholangiocytes, mesenchymal stem cells, and fibroblasts (iPSC-Heps mix [3D]). HFHs and HAHs cultured in static sandwich were used as the control in all experiments. To compare between 2D culture and assembled liver, ANOVA with Wilcoxon test was used: *p < 0.05 (n = 3). Bars in all graphs represent the mean ± SD of three independent experiments. Error bars represent mean ± SD of three experimental experiments.

(F) Characterization of human liver graft entirely regenerated from iPSC-derived cells four days after recellularization, showing homogeneous expression of HNF4α and ALB, but no expression of AFP and ALB was detected. Also, double-immunofluorescence staining for CD31 and CK7 are shown. H&E, hematoxylin and eosin. Hoechst (blue stain) was used as counterstaining.

(G) A1AT production from regenerated liver tissue assembled with human iPSC-human derived cells (assembled human iPSC-liver) in comparison to that from iPSC-Heps cultured alone in static sandwich (iPSC-Heps [3D]) and human iPSC-Heps cultured with iPSC-VECs, iPSC-derived cholangiocytes, mesenchymal stem cells, and fibroblasts (iPSC-Heps mix [3D]). HFHs and HAHs cultured in static sandwich were used as the control in all experiments. Error bars represent mean ± SD of three experimental experiments.