Amelioration of Hyperbilirubinemia in Gunn Rats after Transplantation of Human Induced Pluripotent Stem Cell-Derived Hepatocytes

Abstract

SummaryHepatocyte transplantation has the potential to cure inherited liver diseases, but its application is impeded by a scarcity of donor livers. Therefore, we explored whether transplantation of hepatocyte-like cells (iHeps) differentiated from human induced pluripotent stem cells (iPSCs) could ameliorate inherited liver diseases. iPSCs reprogrammed from human skin fibroblasts were differentiated to iHeps, which were transplanted into livers of uridinediphosphoglucuronate glucuronosyltransferase-1 (UGT1A1)-deficient Gunn rats, a model of Crigler-Najjar syndrome 1 (CN1), where elevated unconjugated bilirubin causes brain injury and death. To promote iHep proliferation, 30% of the recipient liver was X-irradiated before transplantation, and hepatocyte growth factor was expressed. After transplantation, UGT1A1+ iHep clusters constituted 2.5%–7.5% of the preconditioned liver lobe. A decline of serum bilirubin by 30%–60% and biliary excretion of bilirubin glucuronides indicated that transplanted iHeps expressed UGT1A1 activity, a postnatal function of hepatocytes. Therefore, iHeps warrant further exploration as a renewable source of hepatocytes for treating inherited liver diseases.

Generation of Human Liver Chimeric Mice with Hepatocytes from Familial Hypercholesterolemia Induced Pluripotent Stem Cells

“…Moreover, primary FH hepatocytes are difficult to obtain and cannot be expanded in vitro. Although liver repopulation in our chimeric mice is significantly lower than for Fah − / − / Rag2 − / − / Il2rg − / − mice engrafted with pHH (Azuma et al., 2007, Bissig-Choisat et al., 2015), it is comparable with results of recent reports describing NSG mice or Gunn rats (a model of Crigler-Najjar syndrome 1) engrafted with iPSC-derived iHeps (2%–17% [Liu et al., 2011] and 5.1% [Chen et al., 2015]). In the future, crossing our LRG mice with Fah knockout mice could help achieve higher liver chimerism, and thus further upgrade in vivo drug testing studies using engrafted FH iHeps.…”

Methanobactin reverses acute liver failure in a rat model of Wilson disease

“…iPSCs were maintained on Matrigel-coated plates (Corning) in mTeSR cell culture medium and dissociated with 1 U/ml dispase (STEMCELL Technologies) into small clusters and subcultured every 5 to 7 days. WD iPSCs were differentiated into HLCs by a modification of the method reported previously (69,70). iPSCs (5 × 10 4 ) in Matrigel-precoated 6-well plates were incubated in DMEM/ F12 enriched with 100 ng/ml recombinant Activin-A (PeproTech); 100 ng/ml FGF2 (PeproTech); and 50 ng/ml recombinant human WNT3A (R&D Systems).…”

“…iPSCs (5 × 10 4 ) in Matrigel-precoated 6-well plates were incubated in DMEM/ F12 enriched with 100 ng/ml recombinant Activin-A (PeproTech); 100 ng/ml FGF2 (PeproTech); and 50 ng/ml recombinant human WNT3A (R&D Systems). Subsequently, medium was changed up to day 14 according to standard protocols (69,70). Cells were characterized by flow cytometry to assess albumin expression using the anti-human serum albumin Ab (Abcam; catalog ab2406) and the goat anti-rabbit IgG (H+L) secondary Ab with Alexa Fluor 594 conjugate (Thermo Fisher Scientific; catalog A-11012) for detection and by quantitative real-time PCR to assess typical markers of hepatocyte lineage.…”

Biotechnology Challenges to In Vitro Maturation of Hepatic Stem Cells

“…A similar lack of HLC repopulation was reported in Gunn rats, a model for Crigler-Najjar syndrome (deficient in bilirubin metabolism). 132,136 In these experiments, researchers irradiated approximately 30% of the liver area and regeneration was induced by expression of hepatocyte growth factor (HGF) from an adenoviral vector. Transplanted HLCs repopulated 7.5% of the irradiated median liver lobe, quantified by immunostaining, and reduced levels of bilirubin by 60%.…”

“…Transplanted HLCs repopulated 7.5% of the irradiated median liver lobe, quantified by immunostaining, and reduced levels of bilirubin by 60%. 132 …”