| The liver is a complex organ comprised of a variety of cell types: non-parenchymal cells, such as sinusoidal endothelial cells, Kupffer cells and stellate cells, and parenchymal hepatocytes. The hepatocyte is the major functional unit of the liver and is therefore, an appealing area of study. Primary hepatocytes have been used in vitro for toxicology and kinetic studies of drugs. In addition, it has been suggested that hepatocytes be used for transplantation as an alternative to orthotopic liver transplantation, which is currently the only cure for end-stage liver disease but is difficult given the worldwide shortage of donor livers. However, large numbers of hepatocytes that are immunologically matched to the recipient are still needed. Therefore, there is still a need for large numbers of cells for studies.;We first sought to determine if mouse iPSCs were similar to mouse ESCs. We generated two independent iPSC clones by reprogramming mouse embryonic fibroblasts and showed that they were similar to R1 mouse ESCs in their alkaline phosphatase activity, expression of Oct3/4, and their ability to differentiate in vitro into embryoid bodies. To show that mouse iPSCs were truly pluripotent, we conducted tetraploid complementation with transgenic embryos that constitutively expressed enhanced green fluorescent protein (CAG-EGFP). We saw that although extraembryonic tissues expressed EGFP, the embryo proper did not, suggesting that the iPSCs were capable of generating all cell types of the embryo. Furthermore, we showed that the iPS cell-derived liver was normal in morphology, cellular content, and hepatic gene expression when compared with wild-type embryonic liver. These studies demonstrated that mouse iPSCs are similar to mouse ESCs in their developmental competency and can form fetal livers in vivo..;To determine if human iPSC-derived hepatocytes could be used therapeutically, we sought to investigate their repopulation capability in a well-studied model of liver dysfunction. FRGN mice have a global knockout of fumarylacetoacetate hydrolase, which is important for tyrosine catabolism. Knockout of this gene leads to liver failure and death unless the mice are treated with the drug NTBC. These mice also have knockout of Rag2 and Il2rg, resulting in no B, T or NK cells. Furthermore, these mice are bred into the NOD background to deplete macrophages. The complete lack of immune system allows this model to work efficiently with human primary cell transplants. First, we tested whether we were able to repopulate the FRGN liver with primary human hepatocytes. We introduced primary human hepatocytes by intrasplenic injection into adult FRGN mice and showed that although each batch of primary human hepatocytes repopulates with different efficiencies, we were able to highly repopulate a subset of recipients. To test whether iPS cell-derived hepatocytes could be used therapeutically to rescue the mice, we differentiated human iPS cells and transplanted them into adult and neonatal mice at different stages of the differentiation. Although we were able to detect human cells within the parenchyma of FRGN mice, the cells assumed an abnormal morphology within the livers and, in the case of one animal, formed tumors. From our data we concluded that FRGN mice can be repopulated with primary human hepatocytes and that human iPS cell-derived hepatocytes fail to repopulate the damaged mouse liver parenchyma..;One appealing possibility that we have studied is the use of pluripotent stem cells as an unlimited source from which to differentiate hepatocytes to be used in transplantation studies in animal models of liver disease. Embryonic stem cells (ESCs) have the capacity to be propagated indefinitely in culture and are pluripotent because they have the potential to differentiate into many different somatic cell types. Induced pluripotent stem cells (iPSCs) share these characteristics with ESCs, although much work still needs to be done to determine how similar these two cell types are and whether iPSCs truly have the capabilities that ESCs do. The advantage of iPSCs is that they originate from somatic cells, such as skin or blood cells, so they can be easily obtained and, therefore, can be generated from every human being. This provides opportunities to study drug toxicity and metabolism across a wide variety of genetic backgrounds and could one day lead to personalized medicine. In addition, iPSCs derived from patients with metabolic disease may offer a new system to study disease. Furthermore, if hepatocytes differentiated from iPSCs can rescue a mouse model of liver disease, not only would this provide proof-ofprinciple for the therapeutic use of pluripotent stem cells, but it could provide an individualized animal model to study disease and drug toxicity, efficacy, and metabolism. We proposed that hepatocytes differentiated from iPS cells would be competent to repopulate the damaged mouse liver. |
