Human liver chimeric mice carrying human pHH can
Human liver chimeric mice carrying human pHH can facilitate in vivo study of viral hepatitis infections (Bissig et al., 2010; Carpentier et al., 2014) and liver genetic diseases (Bissig-Choisat et al., 2015; Yusa et al., 2011), and the testing of small molecular drug candidates and biological components including phosphodiesterase inhibitors (Legrand et al., 2009). Embryonic stem cell (ESC)- or iPSC-derived iHeps have been used to generate chimeric mice by engrafting them into the liver of non-obese diabetic severe combined immunodeficient (NOD-SCID) mice (Chen et al., 2012), NOD/Lt-SCID/IL-2Rγ (NSG) mice (Liu et al., 2011), or uPA transgenic mice (Basma et al., 2009; Carpentier et al., 2014), where iHeps showed further maturation and regeneration potential and displayed key activities of primary hepatocytes. Our study has produced a mouse model that lacks the gene (Ldlr) involved in a human disease (FH) and is engrafted with patient-specific iHeps to mimic the specific human disease condition and perform in vivo drug testing. This approach has significant advantages over studies using Ldlr knockout mice alone (Ishibashi et al., 1993), as endogenous hepatocytes in the latter mouse model are not responsive to drugs modulating the LDLR pathway. A recent study showed engraftment of FH patient-specific iHeps injected subcutaneously rather than into the liver parenchyma of Rag1/Ldlr mice, but the authors did not perform an assay to demonstrate functional recovery (Ramakrishnan et al., 2015). Another report demonstrated the feasibility of directly engrafting FH (due to compound heterozygosity in LDLR gene) pHH into Fah/Rag2/Il2rg mice, and the phenotype of FH was successfully rescued by LDLR gene therapy using adeno-associated virus (Bissig-Choisat et al., 2015). Because of the lack of Fah, these mice had higher repopulation efficiency (>70%) (Bissig-Choisat et al., 2015) than our mice. However, a problem of this approach is that a population of mouse hepatocytes with intact LDLR remains while the engrafted patients\' pHH did not contain functional LDLR, which can be confounding factors when performing in vivo testing of drugs acting on the LDLR pathway. 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. It should also be considered that mice and humans bear differences in lipoprotein metabolism, and therefore it may be desirable to genetically engineer other mammalian species (e.g., rabbits) (Shiomi and Ito, 2009) for more accurate studies using transplanted human FH iHeps. Nevertheless, despite the physiological differences between mice and humans and the moderate chimerism of our own model, the lowering of plasma LDL-C achieved with LDLR-competent iHeps was significant, while FH iHeps were less effective but exhibited good response to statins and particularly to PCSK9 antibodies. Besides plasma LDL-C levels, we measured endothelial function in aortas of LRG mice to assess the efficacy of engrafted iHeps and these two medications in vivo, as this parameter is affected early in patients with FH (Wiegman et al., 2004). Our results showed that cell therapy and the two medications could improve EDV in chimeric mice, indicating that endothelial function can be used as a parameter to evaluate disease progression during preclinical testing in chimeric LRG mice or other similar animal models. Importantly, we also noticed that the engrafted iHeps could be maintained in LRG mouse liver, and were functional, for at least 3 months. Overall, these results highlight the potential relevance of transplanting iPSC-derived iHeps for the treatment of hereditary metabolic liver disorders (Cantz et al., 2015), and the use of chimeric animals humanized with disease-specific iHeps for preclinical evaluation of novel therapies. However, the long-term therapeutic efficacy of iPSC-based approaches remains unclear, and future studies are necessary to address this as well as safety concerns.