Differentiation Of Human Embryonic Stem Cells Into Hepatocytes

Orthotopic liver transplantation (OLT) remains the most successful treatment for many cases of end-stage liver disease. However, the efficiency of liver transplantation is limited by the shortage of available donor organs, risk of rejection, infections, and other complications caused by the lifelong immunosuppression. Liver cell therapies, including hepatocyte transplantation and bioartificial liver (BAL), are considered promising new approaches to treatment of patients with end-stage liver disease. Unfortunately, The major limitation of these approaches is the availability of human livers as a source of whole liver transplantation or for suspensions of parenchymal cells. With their potential to develop into virtually any cell type, human embryonic stem cells (hES cells) might be an ideal source of donor liver cells for cell therapies aimed at restoring or substituting lost hepatic mass in diseased livers. Nevertheless, there are lots of doubts and uncertainties in the influencing factors and control agents of effectively inducing hES cells differentiation, the efficiency of hES cells'differentiation into hepatocytes, and differentiated cells functional state, etc., and there are many obstacles in application of them clinically. In this study, we investigated the feasibility and methods of differentiation of hES cells into functional hepatocytes, and established a hES cell strain stably transfected by pAlb-EGFP. The studies offered a foundation of gene and stem cell engineering-based regenerative medicine for end-stage liver diseases.Our study mainly includes three parts.1. Differentiation of hES cells into hepatocytes by supplementing soluble growth factors and extracellular matrix.We established a set of standard culture method of hES cells on a feeder layer ofγ-irradiated mouse embryonic fibroblast (MEF) cells.To investigated the differentiated potential of hES cells into the hepatocytes, Firstly, embryoid bodies (EBs) were generated to enhance cell-cell interactions and form the three dimensional structure approaching the embryonic environment. After 7 days, they were inoculated on tissue culture plates coated with collagen type I, and cultured in media containing dexamethasone and insulin. Our results show that the induced cells exhibit similar morphology, phenotype, and functions to hepatocytes.2. Differentiation of hES cells into hepatocytes by co-culture with transgenic stromal cells.In part 1, we demonstrated that hES cells could differentiate into hepatocyte-like cells. However, similar with previously employed methods, this strategy does not exclude the extraembryonic endoderm differentiation of ES cells, which makes the hepatic identities of the differentiated cells controversial. This is especially important for hES cells differentiation, as hES cells tend to differentiate toward extraembryonic endoderm cells, which also express most of the hepatocyte markers, such asα-fetoprotein, albumin and transthyretin. According to the potential of hES cells to develop into virtually any cell type and the mechanism of liver development, a novel strategy has been developed to derive hepatocyes from hES cells using 4 sequential inducing steps lasting 16 days. Firstly, embryoid bodies were generated by growing hES cells in suspension for 2 days; secondly, embryoid bodies were lineage restricted to definitive endoderm with 3 days of treatment with human recombination activin A; thirdly, cells were differentiated further by co-culturing for 5 days with human fetal liver stromal cells (hFLSCs) made transgenic to stably release basic fibroblast growth factor (bFGF); fourthly, treating them for 6 days with soluble signals comprised of hFLSCs-derived bFGF, hepatocyte growth factor, Oncostatin M, and dexamethasone. After treatment of activin A for 3d, more than 80% of the differentiated cells showed expression of a definitive endoderm marker, SOX17 but no AFP expression indicating that these cells were not visceral endoderm cells. Morphologic, phenotypic, and functional analysis proved that hES cells can differentiate into authentic hepatocytes in vitro.3. Establishment of hES cell strain stably transfected with pAlb-EGFP.Although we have developed improvements on strategies to differentiate hES cells into liver fates, the induced cells still a complex, which might be a danger in future application. Albumin (Alb) is expressed both in embryonic and adult liver, which make it as a specifically hepatic marker. In this study, we aimed to generate a hES cell strain expressing enhance green fluorescent protein (EGFP) under the control of the Alb promoter. Because the pAlb-EGFP contained an SV40-driven neomycin selectable marker, which confers resistance to G418 antibiotic, we can select positive clone by G418 conditional culture. The results showed that 200μg/ml is the optimum G418 concentration for selection. Next, we transfected hES cells with pAlb-EGFP by EXGen 500 and selected positive clone with 200μg/ml G418. RT-PCR proved that there is an Alb-EGFP sequence in the gene of positive clone previously selected, which demonstrated the transfection is stable and may be applied in future.Differentiation of hES cells into functional hepatocytes offer a novel method for cell therapies in liver failure. It is of great importance to China, a country with a high morbidity in liver disorders. hES cells, which have permanent self-renewal capability and developmental pluripotency, might be an ideal source of seed cells for BAL, cells transplantation and gene therapy based on liver (stem/progenitor) cells. More importantly, its application in liver dysfunction treatment will be enlarged by nuclear transfer and banking on hES cells. Furthermore, the induction of modern biotechnology will also make a contribution to its clinical application.