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© 2018 The Authors Background & Aims: α1-Antitrypsin deficiency (A1ATD) is an autosomal recessive disorder caused by mutations in the SERPINA1 gene. Individuals with the Z variant (Gly342Lys) retain polymerised protein in the endoplasmic reticulum (ER) of their hepatocytes, predisposing them to liver disease. The concomitant lack of circulating A1AT also causes lung emphysema. Greater insight into the mechanisms that link protein misfolding to liver injury will facilitate the design of novel therapies. Methods: Human-induced pluripotent stem cell (hiPSC)-derived hepatocytes provide a novel approach to interrogate the molecular mechanisms of A1ATD because of their patient-specific genetic architecture and reflection of human physiology. To that end, we utilised patient-specific hiPSC hepatocyte-like cells (ZZ-HLCs) derived from an A1ATD (ZZ) patient, which faithfully recapitulated key aspects of the disease at the molecular and cellular level. Subsequent functional and “omics” comparisons of these cells with their genetically corrected isogenic-line (RR-HLCs) and primary hepatocytes/human tissue enabled identification of new molecular markers and disease signatures. Results: Our studies showed that abnormal A1AT polymer processing (immobilised ER components, reduced luminal protein mobility and disrupted ER cisternae) occurred heterogeneously within hepatocyte populations and was associated with disrupted mitochondrial structure, presence of the oncogenic protein AKR1B10 and two upregulated molecular clusters centred on members of inflammatory (IL-18 and Caspase-4) and unfolded protein response (Calnexin and Calreticulin) pathways. These results were validated in a second patient-specific hiPSC line. Conclusions: Our data identified novel pathways that potentially link the expression of Z A1AT polymers to liver disease. These findings could help pave the way towards identification of new therapeutic targets for the treatment of A1ATD. Lay summary: This study compared the gene expression and protein profiles of healthy liver cells and those affected by the inherited disease α1-antitrypsin deficiency. This approach identified specific factors primarily present in diseased samples which could provide new targets for drug development. This study also demonstrates the interest of using hepatic cells generated from human-induced pluripotent stem cells to model liver disease in vitro for uncovering new mechanisms with clinical relevance.

The studies presented in my thesis delve into the complex relationship between cancer and aging using analytical approaches shared with the field of regenerative medicine. We took advantage of a model of orthotopic cell transplantation to explore the basic question as to whether the contribution of aging to the risk of cancer is, at least in part, related to alterations in the tissue microenvironment. Previous investigations conducted by our research group had revealed that isolated normal hepatocytes form larger clones of daughter cells when transplanted into the liver of old animals as compared to those formed in young recipients. In a relatively simple experiment we then utilized a similar approach to transplant preneoplastic cells isolated from hepatic nodules into either young or old animals of a syngeneic strain. Results were striking: very limited expansion of transplanted nodular hepatocytes was observed in the liver of young animals, while the same cell preparations grew significantly upon injection into older hosts, with a few cases of progression to hepatocellular carcinoma (HCC). The relevance of these findings is twofold. Firstly, they clearly indicate that preneoplastic cell populations isolated from hepatic nodules are not endowed with any measurable degree of growth autonomy, in that they were unable to form nodules when transferred into the liver of young hosts. Thus, their focal growth is dependent on stimuli originating from the local microenvironment. In this respect they behave like normal hepatocytes. Secondly, it is evident from these results that the microenvironment of the aged liver is able to foster the growth transplanted syngeneic nodular hepatocytes, again reproducing analogous findings already obtained with normal cells. Having established this fundamental fact, it became important to explore possible biological and underlying molecular mechanisms explaining the growth-promoting nature of the aged liver microenvironment. To this end, intriguing insights came again from the field of regenerative medicine. Our research group is involved in the characterization of an experimental model of massive liver repopulation through hepatocyte transplantation. This approach was set up in our laboratory and is based on the administration of retrorsine (RS), a naturally occurring alkaloid, as a preconditioning treatment. Rat liver exposed to RS and then transplanted with isolated normal hepatocytes is entirely replaced by donor-derived cells within a few months. Importantly, transplanted nodular hepatocytes can also selectively expand in the RS-treated liver, rapidly evolving to HCC. Again, neither normal, nor nodular hepatocytes are able to proliferate when injected into the liver of untreated recipients, as already noted above. This pattern of findings is similar to that observed in the aged liver, although the magnitude of the observed phenomena is far greater following exposure to RS. Moreover, it is apparent from the foregoing discussion that the growth of normal and nodular hepatocytes is controlled by fundamentally similar biological mechanisms, such that carcinogenesis and liver repopulation represent in fact “two sides of the same coin”, as it has been proposed. Given the similarities of biological responses between the RS-treated and the aged liver, we then probed into possible cellular and molecular analogies shared by the two systems. Much to our surprise, it was found that exposure to RS is a powerful inducer of cell senescence in hepatocytes in vivo. As reported in the preceding section of this report, several markers of cell senescence were expressed by RS-exposed hepatocytes, including senescence-associated β-galactosidase (SA-β-gal), cell hypertrophy, a persistent block in the cell cycle coupled with activation of cell growth pathways. The picture emerging from these findings is that RS treatment, which sets the stage for the selective growth of both normal and nodular transplanted hepatocytes, leading to massive liver repopulation and emergence of HCC, respectively, is also associated with the induction of a senescent phenotype in resident hepatocytes. Such a senescence phenotype is commonly found in aged tissues including the liver, as also shown in our studies. This phenotype is likely to explain, at least in part, the similarities observed between the microenvironments of the aged liver and that of RS-exposed liver. The last section of this thesis describes studies aimed at reversing the RS-induced alterations in the liver microenvironment and, in doing so, at verifying whether this would modulate the growth of preneoplastic lesions promoted by RS. Animals given the genotoxic agent diethylnitrosamine (DENA) followed by RS developed large hepatocyte nodules within a few months. However, the growth of preneoplastic nodules was significantly delayed in animals receiving hepatocyte transplantation following the DENA+RS regimen. These results represent a proof of principle that an altered microenvironment is indeed a powerful driving force in neoplastic progression; most importantly, they clearly indicate that strategies aimed at “normalizing” such an altered microenvironment might impact on the rate of progression of the neoplastic process.

Summary Use of hepatocytes derived from induced pluripotent stem cells (i-Heps) is limited by their functional differences in comparison with primary cells. Extracellular niche factors likely play a critical role in bridging this gap. Using image-based characterization (high content analysis; HCA) of freshly isolated hepatocytes from 17 human donors, we devised and validated an algorithm (Hepatocyte Likeness Index; HLI) for comparing the hepatic properties of cells against a physiological gold standard. The HLI was then applied in a targeted screen of extracellular niche factors to identify substrates driving i-Heps closer to the standard. Laminin 411, the top hit, was validated in two additional induced pluripotent stem cell (iPSC) lines, primary tissue, and an in vitro model of α1-antitrypsin deficiency. Cumulatively, these data provide a reference method to control and screen for i-Hep differentiation, identify Laminin 411 as a key niche protein, and underscore the importance of combining substrates, soluble factors, and HCA when developing iPSC applications., Highlights • iPSC-derived hepatocytes (i-Heps) are functionally limited compared with primary cells • Factors within the extracellular niche likely play a role in bridging this gap • Laminin 411 was shown to be an important niche factor for i-Heps • High content image analysis (HCA) can help development of i-Hep applications, Rashid and colleagues demonstrate the utility of a high-throughput imaging platform for identification of physiologically relevant extracellular niche factors to advance i-Heps closer to their primary tissue counterparts. The extracellular matrix (ECM) protein screen identified Laminin 411 as an important niche factor facilitating i-Hep-based disease modeling in vitro.