In vivo liver regeneration potential of human induced pluripotent stem cells from diverse origins

Abstract

Human induced pluripotent stem cells (iPSCs) are a potential source of hepatocytes for liver transplantation to treat end-stage liver disease. In vitro differentiation of human iPSCs into hepatic cells has been achieved using a multistage differentiation protocol, but whether these cells are functional and capable of engrafting and regenerating diseased liver tissue is not clear. We show that human iPSC-derived hepatic cells at various differentiation stages can engraft the liver in a mouse transplantation model. Using the same differentiation and transplantation protocols, we also assessed the ability of human iPSCs derived from each of the three developmental germ layer tissues (that is, ectoderm, mesoderm, and endoderm) to regenerate mouse liver. These iPSC lines, with similar but distinct global DNA methylation patterns, differentiated into multistage hepatic cells with an efficiency similar to that of human embryonic stem cells. Human hepatic cells at various differentiation stages derived from iPSC lines of different origins successfully repopulated the liver tissue of mice with liver cirrhosis. They also secreted human-specific liver proteins into mouse blood at concentrations comparable to that of proteins secreted by human primary hepatocytes. Our results demonstrate the engraftment and liver regenerative capabilities of human iPSC-derived multistage hepatic cells in vivo and suggest that human iPSCs of distinct origins and regardless of their parental epigenetic memory can efficiently differentiate along the hepatic lineage.

Fig. 1

DNA methylation analysis of human iPSCs, their parental cells, and ESCs. (A) Multidimensional scaling plot showing the relationships among all individual human iPSC lines analyzed in our study. Euclidian distance and the top 5% most variable autosomal loci across all samples analyzed were used. Color code for each group of cell lines analyzed is shown in the figure. iK1, iK2, and iK3 are iPSC lines derived from adult human keratinocytes (Kera). iM2, iM3, and iM7 are iPSC lines derived from human bone marrow mesenchymal stem cells (MSCs). iLC1 and iLC2 are iPSC lines derived from adult human fibroblasts (PLCF). iH11, iH14, and iH10 are from iPSC lines derived from primary human hepatocytes (Hep). Both H1 and H9 are human ESC lines. Mes, mesoderm; Ect, ectoderm; End, endoderm. (B) Hierarchical clustering using Euclidian distance and top 5% most variable autosomal loci across all analyzed samples. Color bars on the side identify the different sample groups. The color code expresses the log₂ methylation ratio. (C) Venn diagrams showing the number of autosomal loci differentially methylated between each iPSC group and the corresponding parental cells, as obtained from our linear model analysis (adjusted P < 0.001). Some of the representative genes associated with the differentially methylated features, based on functional gene set enrichment analysis, are listed for both common and distinct changes occurring during reprogramming among different source-derived iPSCs (hypermethylated, red; hypomethylated, blue).

Fig. 2

Comparison of directed hepatic differentiation in vitro among iPSC lines from different developmental origins. (A) Steps along the differentiation pathway from human iPSCs to hepatocyte-like cells. The key factors that are responsible for directing each stage of differentiation and the time course of commitment to the hepatic lineage are shown. Stage-specific markers including genes and surface phenotypes are indicated under the cells at each differentiation stage. (B to D) Using this protocol, all human ESCs and human iPSCs of different origins were able to differentiate into (B) DE (definitive endoderm, SSEA3^- CXCR4⁺), (C) HP (hepatic progenitors, AFP⁺, red), and (D) MH (mature hepatocyte-like cells, ALB⁺ green, and AAT⁺ red) with a comparable efficiency to one another. Representative results are shown with the H1 human ESC line and human iPSC lines derived from each germ layer tissue. Magnification, ×100. Scale bar, 100 μm. (E and F) All of these human ESC- and iPSC-derived MH cells display cytochrome P450 (CYP450) metabolism (E) and albumin (ALB) secretion (F) (n = 3 experiments, means ± SEM). Representative results are shown for the H1 human ESC line and one of each germ layer tissue–derived human iPSC lines. Day 20 MH cells were incubated with CYP3A4 pGlo substrates; at 3 hours after treatment, 50 μl of culture medium was removed and read on a luminometer. CYP3A4 activity is expressed as relative luminescence units (RLU)/ml of culture medium (n = 6 experiments). All iPSC lines exhibited similar amounts of CYP450 enzyme activities when compared to primary hepatocytes (PHs) and human ESCs. Human ALB secretion in hepatic differentiation culture (F). The cultured medium of human ESC- or iPSC-derived day 20 MH cells (∼1 million/ml for 2 days) was analyzed using human ALB ELISA assays.

Fig. 3

In vivo engraftment and regeneration potential of hepatic cells derived from human iPSCs and ESCs. (A) Diagram depicts liver injury model, transplantation protocol, and analyses used for the in vivo study. (B) Survival rates for NSG mice treated with DMN for 4 weeks that were given human iPSC-derived hepatic cells compared to control mice that did not receive donor cells. The day of transplantation is day 0. Transplanted mice (n = 16 to 20 per group) showed higher survival rates when compared with control mice (n = 17). *P < 0.05, compared with control mice by a log-rank test. (C) Healthy normal (NL) mice, 4-week DMN-treated mice that did not receive human cells, and mice that received human PH cells were used as negative and positive controls, respectively. The intraperitoneal injection of DMN caused hepatic fibrosis, which was reflected by the disruption of tissue architecture and the marked increase in fibrosis in DMN-injected mouse liver. Neutrophils and mononuclear cells infiltrated the livers of the DMN-treated mice (DMN control). Magnification, ×100; scale bar,100 μm. (D to F) CYP2E1 (red) and ALB (green) costaining (yellow) images of DMN-treated NSG mouse liver 8 weeks after intravenous transplantation with human ESC- or iPSC-derived (D) day 5 DE cells (2 × 10⁶), (E) day 10 HP cells (2 × 10⁶), and (F) day 20 MH cells (2 × 10⁶). Representative images of hepatic cells derived from ESCs (H1 and H9) and iPSCs originating from each germ layer are shown.

Fig. 4

In vivo engraftment efficiency of human iPSC-derived multistage hepatic cells. (A) Large area scan images of human albumin (ALB, green) staining in mouse liver transplanted with 2 × 10⁶ DE cells derived from human iPSCs of different origins and from human ESCs. Liver images were taken using the motorized Nikon Ti-E microscope with an Encoded Motorized XY stage and a function in NIS-Elements to generate these montage images. Magnification, ×200; scale bar, 100 μm. (B) The engraftment percentages in mouse liver for multistage hepatic cells derived from human iPSCs (iH6-14) were calculated on the basis of human-specific ALB-positive staining and the large area scan images of multiple different liver lobes and lobules obtained from the recipient NSG mice 8 weeks after transplantation of 0.1 to 1 × 10⁶ cells (n = 3 to 10 mice, means ± SEM). *P < 0.05 by Mann-Whitney test. Similar results were observed with human ESCs and human iPSCs from different tissue origins. (C) The engraftment percentages in mouse liver for multistage hepatic cells derived from diverse origin human iPSCs were calculated on the basis of human ALB-positive staining and the large-area scan images of multiple different liver lobes and lobules obtained from the recipient NSG mice 8 weeks after transplantation with 2 × 10⁶ cells (n = 3 to 12 mice, means ± SEM). (D) An increased percentage of human ALB-positive cells was detected in mouse liver when a higher dose (7 × 10⁶) of human iPSC-derived DE cells was transplanted. Magnification, ×200; scale bar, 100 μm.