Title: Hepatocytes and liver-specific non-parenchymal cells by differentiation from human pluripotent stem cells
Other Titles: Hepatocyten en leverspecifieke niet-parenchymale cellen door differentiatie van menselijke pluripotente stamcellen
Authors: Roelandt, Philip
Issue Date: 17-Nov-2011
Abstract: The liver is the most important metabolic organ and has multiple synthetic and detoxifying functions, including production of proteins, storage, metabolization and detoxification. The liver contains aside from the 60% hepatocytes also non-parenchymal liver-specific cells, including Hepatic Stellate Cells (HSC) and Liver Sinusoidal Endothelial Cells (LSEC). HSC are important for the storage of vitamin A, and in the pathogenesis of fibrosis and cirrhosis, while LSEC are important for the production of coagulation factor VIII and scavenger function of multiple nutrients. One of the most striking features of the liver is its enormous regeneration capacity. Following partial hepatectomy or minor damage, mature hepatocytes re-enter cell cycle and regenerate the lost parenchyma. When the damage is more severe and/or prolonged, facultative liver progenitor cells in the periportal zone proliferate and generate new hepatocytes. If this mechanism is still insufficient, acute liver failure occurs. In many cases, urgent liver transplantation is the only solution, but this option is limited by donor organ shortage. Aside from the limited availability of hepatocytes for therapeutic purposes, shortage of human hepatocytes also precludes their widespread use in the pharmaceutical industry to test drug toxicity and drug metabolization, or develop new anti-hepatitis virus drugs. Therefore many groups have started to evaluate possible renewable sources of hepatocytes.Sources that are being considered include pluripotent embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC), created by introducing transcription factors that reprogram adult cells into ESC-like cells. In the Stem Cell Institute, rat multipotent adult progenitor cells (MAPC) were already successfully differentiated into chiefly immature hepatocyte-like cells. In this thesis we evaluated whether the protocol developed for rat MAPC could be used to generate mature functional hepatocytes from hESC and hiPSC. (1) To test if the hepatic differentiation protocol could be used to generate human hepatocytes and liver-specific non-parenchymal cellsWe first evaluated if the existing rodent protocol also allows generation of functional hepatocytes from human embryonic and induced pluripotent stem cells. The progeny was extensively characterized on gene expression, protein synthesis and functional capacities compared to human primary hepatocytes. Secondly we evaluated if liver-specific non-parenchymal cells were also present in the final progeny. We have demonstrated that it is possible to generate functionally active hepatocytes from hESC and iPSC, as well as hepatic stellate cells (HSC) and liver sinusoidal endothelial cells (LSEC). The differentiation is cytokine-dependent as significantly lower gene expression and functional capacities were noted when the differentiation was performed in the absence of cytokines. (2) To optimize the hepatic differentiation protocolEvery step of the differentiation protocol developed for rat MAPC was re-evaluated to determine whether the addition of the different cytokines was necessary. The initial four step protocol contained serum and 10 cytokines namely Activin-A and Wnt3a (step I), BMP4 and FGF2 (step II), FGF1, FGF4 and FGF8b (step III) and HGF and Follistatin (step IV). The new optimized protocol to stepwise commit hESC to hepatocytes and non-parenchymal liver cells results in at least the same degree of phenotypic and functional hepatocyte generation. The optimized protocol only contains 5 cytokines and differentiation is possible in the absence of bovine serum, making it less expensive, and more readily translatable to the clinic. Activin-A and Wnt3a were shown to be essential for primitive streak/mesendoderm/definitive endoderm formation, while BMP4 was crucial for hepatic endoderm specification. FGF2, FGF4, FGF8b and Follistatin could be omitted from the protocol without negative influence on the final gene expression and functional capacities of hESC progeny. Dexamethasone was shown to be crucial for hepatocyte maturation, while prolongation of the final step also increased hepatocyte maturation. Addition of Oncostatin M or Wnt3a during step IV did not improve differentiation. Co-culture with endothelial progenitor cells might be beneficial for hepatocyte maturation, as a single addition of BOECs to the hESC progeny improves hepatocyte gene expression. (3) To develop methods to enrich for different cell fractionsSince the final progeny is still mixed, we aimed to develop methods to isolate the different cell fractions of interest. We demonstrated that it is possible to generate (semi)single cell suspensions of hESC progeny. As hepatocytes are large, isolation using FACS is difficult; however, we demonstrated that enrichment for hepatocytes is possible using immunomagnetic sorting for ASGR1+ cells. Unfortunately, cells other than hepatocytes are also selected, yielding increased hepatocyte purity, which is still incomplete. Preliminary studies suggests that HSC and (LS)EC may also be enriched from the hESC progeny using FACS with respectively UV fluorescence/Bodipy (HSC) or antibodies against endothelial surface proteins. (4) To determine if hESC/iPSC hepatic progeny can be infected by hepatitis C virus (HCV)We hypothesized that hESC/iPSC derived hepatic progeny could support HCV infection and could provide better insights in the HCV replication cycle. We demonstrated that the receptors for HCV entry are present on hESC hepatic progeny and that following inoculation of HCV, hepatic progeny secreted albumin, ApoB100 and HCV virions, which could re-infect Huh7.5.1 cells. The HCV RNA replication could be inhibited by both NS3/4A protease inhibitor and non-nucleosidic NS5B polymerase-inhibitor. Based on RT-qPCR, immunofluorescence and functional analysis, we estimate that 5% of the hESC/hiPSC-progeny is susceptible to HCV infection.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Hepatology
Interdepartemental Stem Cell Institute (-)

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