Inherited Metabolic Disorders of the Liver
Inherited metabolic disorders (IMD) include more than 70 monogenetic disorders that are mostly associated with enzyme deficiencies. Although IMDs are rarely diagnosed, their combined incidence among full-term neonates has been estimated to account for 20 % of illnesses. One of the most common IMDs originating in the liver and necessitating transplantation is α1-antitrypsin deficiency. Although several in vitro and in vivo models have been used to study this disease, the application of patient-specific hIPSCs and their differentiation to hepatic lineages has allowed not only physiologically relevant insights into the disease mechanisms but also provides the opportunity to understand patient-patient variabilities in phenotypes and sets the stage for the identification of novel biomarkers and the design of drugs that target them. This chapter discusses the wide application range of hIPSCs and addresses how this cellular system can meet previous shortcomings in advancing research into the molecular mechanisms of disease.
KeywordsLiver disease Cirrhosis hIPSC-derived hepatocytes Alpha-1-antitrypsin deficiency
7.1 Introduction: Inherited Metabolic Disorders of the Liver
The liver is a unique and vital organ by the broad spectrum of its functions. It processes consumed substances and sustains reserves of iron, vitamins, and minerals. It detoxifies alcohol, drugs, and other chemicals and removes inhaled poisons such as exhaust or smoke, all of which accumulate in the bloodstream. The liver is also the site of synthesis of plasma proteins such as albumin, blood clotting factors, hormones, as well as bile required for the digestion of lipids. Finally, the liver has an essential metabolic activity by storing glycogen. Most of these activities are managed by one cell type, the hepatocyte, which constitutes the main cellular unit of the liver (Kmieć 2001). Genetic mutations targeting any gene involved in the functioning of hepatocytes can destroy the capacity of the liver to carry out these essential activities, thereby threatening the life of the afflicted patient.
Genetic diseases associated with impaired liver function are combined under the umbrella term “inherited metabolic disorders” (IMDs) which was originally coined “inborn errors of metabolism” by the British physician Archibald Garrod (1923). This class of heterogeneous diseases includes almost 70 disorders that are characterized primarily by defects in single, enzyme-coding genes that lead to lack of enzymatic activity and toxic substrate accumulation (Martins 1999). While individual events of IMDs are relatively rare, taken together they account for an incidence of 1 in 2,500–5,000 among full-term neonates (Seymour et al. 1997). Most of these disorders require medical treatment, which needs to be employed immediately after birth to avoid interference with early mental and physical development. IMDs that compromise vital liver functions are generally targeted with orthotopic liver transplantation (OLT) since only a healthy donor liver can restore the missing metabolic function. However, this intervention is associated with drawbacks since it implies high risk of surgical complications, indefinite immunosuppression associated with severe side effects, and potential organ rejection. Furthermore, the number of organ donors has remained constant during the past 10 years, while the demand for liver transplantation has more than doubled in the meantime. This situation is anticipated to worsen in the foreseeable future due to the hepatitis C pandemic and increase in cirrhosis associated with obesity. The development of alternative therapies to OLT has therefore become a major objective in the field of regenerative medicine. Further goals for the translational research community include the advancement of physiologically relevant cell models to study the underlying disease mechanisms of the respective disorders and the design and screening of novel drug compounds.
7.2 An Example: α1-Antitrypsin Deficiency
α1-antitrypsin (A1AT) deficiency is one of the most common IMDs, affecting 1 in 2000 individuals of North Caucasian decent (Sveger 1976). A1AT deficiency is characterized by a point mutation in the A1AT gene (SERPINA1), which is primarily expressed and synthesized by hepatocytes and secreted as a circulating plasma glycoprotein. While wild-type A1AT acts as an antiprotease that enzymatically inhibits overactive neutrophil elastase in pulmonary tissues, defective A1AT protein synthesis results in intracellular A1AT protein polymerization, accumulation and reduced A1AT levels in the circulation and in target organ tissues such as the lungs. A1AT deficiency is inherited as an autosomal codominant trait leading to homozygous or heterozygous phenotypes. Homozygous A1AT-deficient individuals are at an increased risk of developing chronic hepatitis, liver cirrhosis, and chronic-obstructive respiratory disease. This chapter introduces the pathophysiology of A1AT deficiency and explains the suitability of human-induced pluripotent stem cells (hIPSCs) to study this disease and unearth unknown mechanisms involved in its pathogenesis.
7.2.2 Genetic Basis, Molecular Mechanisms, and Epidemiology
7.2.3 Clinical Symptoms
The significant reduction of secreted A1AT is caused by pathogenic accumulation of A1AT in the liver of homozygous PiZZ individuals (Laurell and Eriksson 1963). This leads to toxic gain-of-function mechanisms in hepatocytes and marks A1AT deficiency as the most common genetic liver disease affecting children and requiring juvenile liver transplantation (Gartner Jr et al. 1984). A1AT deficiency can present as neonatal cholestasis (jaundice), juvenile hepatitis, enlarged spleen, and swelling of the abdomen during the first months or years of life. 10–15 % of A1AT-deficient children and 40–60 % of overall PiZZ patients will develop chronic liver disease at some stage, which may advance to liver cirrhosis or liver cancer (Sveger 1976, 1988; Eriksson et al. 1986).
Similar to lung disease, there is great variability between A1AT-deficient patients in the phenotypic expression of associated liver disease. The onset, progression, and severity of liver disease have been shown to vary widely even between PiZZ siblings (Hinds et al. 2006). Generally, higher rates of degradation of misfolded intracellular A1AT polymers have been linked with reduced liver disease burden (Wu et al. 1994). As such, mechanisms to upregulate autophagy have been tied to reduced A1AT polymer concentrations and less liver injury in a mouse model (Kaushal et al. 2010). To date, the only curative intervention for end-stage A1AT deficiency is whole-organ liver transplantation (Fairbanks and Tavill 2008). The overall mortality of individuals with severe A1AT deficiency has been examined in several studies and has been reported between 18 and 37 % (Larsson 1978; Seersholm et al. 1994; Stoller et al. 2005).
Until recently, the detailed molecular mechanisms causing Z A1AT polymer accumulation in the ER lumen of hepatocytes to trigger liver disease were not well understood. Improved insights into these processes would improve the understanding of what distinguishes the A1AT-deficient subpopulation susceptible to liver disease (~20 %) from those protected (~80 %) (Fairbanks and Tavill 2008). Given that the mere Glu342Lys amino acid substitution in SERPINA1 cannot account for the emergence of liver disease, PiZZ individuals without liver disease likely carry certain genetic traits that protect from A1AT polymer-associated disease. Alternatively, PiZZ individuals with liver disease may be exposed to environmental factors or carry a specific genetic deposition that renders them susceptible to disease. Several groups have attributed the buildup of A1AT aggregates and resultant liver injury to impaired and delayed degradation of Z A1AT (Wu et al. 1994). This may be due to compromised interactions between Z A1AT and factors of the calnexin/calreticulin cycle, which generally aid glycoproteins in their folding and tag permanently misfolded glycoproteins for proteasomal degradation (Cameron et al. 2009; Qu et al. 1996). Besides impaired proteasomal degradation of Z A1AT molecules, elimination of A1AT polymers via autophagy may also be reduced, delayed, or overwhelmed (Perlmutter 2011). Other studies have shown that A1AT polymer accumulation activates the NF-kB pathway independent of the unfolded protein response (UPR), triggering toxic ER stress signals in protein-overloaded hepatocytes (Hidvegi et al. 2005; Ordóñez et al. 2013). The absence of the UPR may prevent downstream pathways from eliminating misfolded proteins and appropriately regulate apoptosis under chronic ER stress (Hidvegi et al. 2005). It also has been proposed that A1AT polymer-bearing hepatocytes cannot recognize the ordered structures of A1AT polymers as misfolded proteins and therefore fail to mount strong protein disposal pathways (Graham et al. 1990; Hidvegi et al. 2005).
7.3 In Vitro and In Vivo Models: How to Study α1-Antitrypsin Deficiency
As outlined in the previous sections, A1AT deficiency is a liver disease with substantial health consequences but limited options for treatment. In the past, the pressing need to gain insights into the underlying disease mechanisms and the development of new therapeutic tools has been hampered by limited availability and viability of primary A1AT-deficient and wild-type hepatocytes to study the disease. Although animal models have contributed to improved understanding of A1AT deficiency, physiological variability between animal and man has questioned the extrapolation of findings into a Homo sapiens context. The advent of human-induced pluripotent stem cells (hIPSCs) derived from patients with A1AT deficiency and their differentiation to the hepatic lineage has generated cells which phenocopy the disease phenotypes and can target these aforementioned obstacles (Rashid et al. 2010). This section discusses the use and shortcomings of alternative A1AT deficiency models and how hIPSC-derived hepatocytes have developed into a suitable platform for investigating the pathophysiology of A1AT deficiency.
7.3.1 Primary PiZZ Hepatocytes
Primary hepatocytes isolated from PiZZ patients are the most appropriate cell model to study A1AT deficiency. However, it requires invasive biopsies to access the liver tissue. Furthermore, multiple groups have failed to extract and plate high-quality PiZZ hepatocytes from cirrhotic liver tissue which can become available during liver transplantations (Gramignoli et al. 2013). At this stage of disease severity, PiZZ hepatocytes are generally incorporated into a highly fibrotic tissue and are therefore very difficult to isolate, let alone culture (Bhogal et al. 2011). At earlier stages of disease when the liver is still pre-fibrotic, isolation of PiZZ hepatocytes through enzymatic digestion of liver explant tissue may be more feasible. However, oftentimes PiZZ patients are asymptomatic and therefore remain undiagnosed at that point, making it difficult to obtain biological material. Furthermore, due to the absence of long-term culture systems for primary hepatocytes, the study of disease progression over time cannot be accomplished. Finally, the dissociation of hepatocytes from their niche liver microenvironment and subsequent plating in 2D conditions is well known to cause loss of hepatic functionality in primary hepatocytes (Bhatia et al. 2014; Gramignoli et al. 2013).
7.3.2 Evaluation of Alternative Models to Study α1-Antitrypsin Deficiency
In the past, several in vitro and in vivo models have aided in advancing the A1AT deficiency field. For example, cloning of human PiM A1AT into the murine germ line generated transgenic mice that expressed human A1AT levels in serum and liver (Carlson et al. 1988; Sifers et al. 1987). Using the same technique, Carlson et al. and others generated transgenic mice expressing the human PiZ A1AT gene product. These animals exhibited mutant A1AT synthesis and showed retention of mutant A1AT in hepatic ER, hepatocellular damage, and chronic liver injury that correlated with the level of hepatic PiZ A1AT accumulation. The phenotypes in pups resembled human neonatal hepatitis (Carlson et al. 1989; Dycaico et al. 1988). Despite the significant increase in inflammation recorded in these transgenic PiZ mice, there was no statistically significant increase in fibrosis. This was attributed to the faster metabolism and shorter life expectancy of mouse versus man, which may not suffice for the development of chronic disease, including fibrosis, cirrhosis, and hepatocellular carcinoma.
In order to evaluate the pathogenic impact of an elastase to antielastase imbalance on pulmonary health, the pallid mouse has been used to study A1AT deficiency (Martorana et al. 1993). This mouse strain suffers from hereditary emphysema and develops pulmonary lesions comparable with human emphysema. While the hepatic A1AT activity is not disrupted in this mouse model, circulating A1AT levels are reduced and exhibit a diminished capacity for elastase inhibition. Despite the suitability of studying the pallid mouse for its reduced serum elastase inhibitory effects and resultant lung tissue damage, it cannot provide insight into the underlying mechanisms of hepatic disease in PiZZ A1AT humans (Martorana et al. 1993).
Several other yeast and mammalian cell line models have also been shown to recapitulate features of A1AT deficiency (Gosai et al. 2010; Wu et al. 1994; Ordóñez et al. 2013; Cabezn et al. 1984; Kruse et al. 2006; McCracken and Brodsky 1996; Scott et al. 2007; Werner et al. 1996). Despite the exciting findings obtained from these studies, the lack of physiological human A1AT expression makes it difficult to draw conclusions into the pathogenesis of A1AT deficiency. Multiple A1AT gene copy numbers, interference of endogenous animal antiproteases, a genetic background that may not permit onset of disease upon A1AT polymer accumulation, and the absence of endogenous promoters and transcription factors to activate A1AT expression all contribute to a likely non-physiological system to study A1AT deficiency. Furthermore, differences in tissue composition, anatomy, physiology, lack of conservation of gene order, and the animal’s inbred genetic background can direct SERPINA1 mutations to phenotypes that may not faithfully phenocopy the clinical picture of human A1AT deficiency (Shapiro 2007). For instance, instead of a single gene, as observed in primates, murine SERPINA1 exists as a small gene family of up to five genes that synthesize several isoforms (Borriello and Krauter 1991). It still remains to be determined which member/s of this gene cluster correspond to the functional equivalent of human A1AT (Barbour et al. 2002).
7.4 Modeling α1-Antitrypsin Deficiency Using Human-Induced Pluripotent Stem Cells
7.4.1 Human-Induced Pluripotent Stem Cells
hIPSCs, derived from patients with specific disease backgrounds and their subsequent differentiation to specialized somatic cells in which the disease manifests, have emerged as in vitro systems to model various diseases ex vivo. The derivation of hIPSCs followed shortly after the very first induced pluripotent stem cell lines were generated from mouse embryonic fibroblasts (Takahashi et al. 2007; Takahashi and Yamanaka 2006). This was achieved through forced expression of transcription factors c-Myc, Oct3/4, Sox2, and Klf-4 (Yamanaka factors) in fibroblasts. While this process of direct reprogramming was previously performed with viral vectors, several techniques are now available that use non-integrative methods to safely deliver the reprogramming factors (e.g., episomal vectors) (Yu et al. 2011). hIPSCs resemble embryonic stem cells (pluripotent stem cells derived from an embryo at the blastocyst stage) in terms of their expression of pluripotent genes, DNA methylation patterns, doubling time, formation of embryoid bodies, teratoma formation, and viable chimeras, as well as their potency for self-renewal and differentiation into almost any cell type (Wernig et al. 2007). hIPSC research and the development of differentiation protocols to generate specific cell types have promised to yield great advancements for the disease modeling field but also for developmental biology, personalized cell-based therapy, and the generation of platforms to develop and screen new pharmaceuticals (Rashid and Vallier 2010). In the field of transplantation and regenerative medicine, the possibility to produce and implant personalized hIPSC-derived somatic cells into patients removes the risk of tissue rejection inherent to allogeneic transplants, high costs associated with invasive transplantation, and lifelong administration of immunosuppressive drugs and bypasses some of the ethical issues associated with embryonic stem cells.
7.4.2 Derivation and Hepatic Differentiation of α1-Antitrypsin-Deficient hIPSC
In addition, A1AT-ZZs exhibited all disease-specific characteristics of A1AT deficiency, including A1AT polymer formation, intracellular retention in the hepatic ER, and reduced A1AT secretion (Rashid et al. 2010; Tafaleng et al. 2015; Wilson et al. 2015). Thus, A1AT-ZZ hepatocyte-like cells represent an attractive system to study the molecular mechanisms directing the disease pathophysiology.
7.5 Applications of α1-Antitrypsin-Deficient hIPSC-Derived Hepatocytes
7.5.1 Cell Therapy: Genetic Correction of Diseased hIPSCs to Generate Wild-Type hIPSC
In order to generate a wild-type cell line, our group employed zinc finger nucleases and piggyBac-based genome-editing techniques to correct the SERPINA1 mutation in hIPSCs responsible for A1AT deficiency, creating an otherwise isogenic wild-type hIPSC line (Yusa et al. 2011). This cell line was termed A1AT-RR, referring to the corrected SERPINA1 gene. Once differentiated to hepatic lineages, the disease phenotype was rescued in this cell type and A1AT-RRs exhibited normal levels of A1AT secretion as well as the absence of intracellular A1AT polymer formation or accumulation (Yusa et al. 2011). The resulting wild-type hIPSC-derived hepatocytes were transplanted successfully into animal models for liver failure, providing a first proof of principle that hIPSCs could be used for personalized medicine of inherited metabolic diseases (Yusa et al. 2011). With increased hepatic maturity, we anticipate that transplantation of these cells will be able to correct hepatic functionality.
7.5.2 Disease Modeling: Linking Hepatic Accumulation of α1-Antitrypsin Polymers to Pathological Liver Conditions
Despite the volume of information available about the intracellular polymerization of A1AT, it has been unclear how the A1AT polymer retention leads to liver disease. Due to the capacity of A1AT-deficient hIPSC-derived hepatocytes to be cultured long-term, it is possible to observe the accumulation of A1AT polymers over time. Studies aimed at investigating the functional divergences between diseased and healthy hepatocytes can utilize A1AT-ZZs and A1AT-RRs, which only differ in the absence or presence of the SERPINA1 mutation. This allows for detailed comparisons of genetic and protein material at modeled end-stage liver disease, circumventing the interference of differing genetic backgrounds. Using this cellular platform, we have been able to dissect potential pathways involved in linking polymer accumulation and liver disease and allowed us to detect novel biomarkers for this disease (Segeritz et al. in preparation). Interestingly, recent genome-wide studies have also revealed novel aspects of the disease including UPR activity early during the process of hepatocyte specification (Wilson et al. 2015). Thus, these data which would have been impossible to obtain in any other model system suggest that liver disease associated with A1AT could be induced during early liver development.
7.5.3 Patient-Patient Variability: Differentiating the Susceptible from the Protected
hIPSC-derived hepatocytes derived from A1AT-deficient patients have also aided in addressing the question of why some affected individuals are protected from acquiring liver disease, while others suffer from cirrhotic livers that require liver transplantation. The derivation of hIPSCs from A1AT-deficient patients with and without disease and their differentiation to hepatocyte-like cells has allowed insights into the individual disease phenotypes (Tafaleng et al. 2015). While the intracellular Z A1AT in hIPSC-derived hepatocytes of patients without liver disease was primarily degraded by autophagic pathways, elimination of Z A1AT was delayed in hIPSC-derived hepatocytes of patients that suffered from liver disease. Z A1AT in the latter group also appeared to accumulate in ER inclusions. This work suggested that the observed variation in liver disease phenotypes of A1AT deficiency is linked to the rate of misfolded A1AT degradation. This opens the door for the development and testing of specific drug targets that control proteostasis and enhance intracellular degradation such as carbamazepine (Hidvegi et al. 2010). Furthermore, these observations also indicate that hIPSC-derived hepatocytes can be used to predict disease progression and susceptibility, allowing for the implementation of personalized therapeutics.
7.5.4 Drug Development and Drug Screening: Finding a Cure for α1-Antitrypsin Deficiency
Due to the current absence of therapies for the treatment of A1AT deficiency, there is great urgency to develop and test new drug compounds in human systems. hIPSCs derived from A1AT-deficient individuals and differentiated to hepatocytes could act as a suitable platform to screen efficacy of novel drug compounds. Previously, hIPSC-derived hepatocytes have been employed in drug toxicity studies and to show patient-specific sensitivity to a given drug compound (Cayo et al. 2012; Takayama et al. 2013). However, for the first time, a recent separate study has shown that carbamazepine could improve autophagic flux in A1AT-ZZ hepatocyte-like cells (Wilson et al. 2015), thereby reinforcing the interest of clinical trial currently testing this small molecule for A1AT deficiency. Considered together these results exemplify how patient-specific, hIPSC-derived hepatocytes could be used to screen drug compounds to reverse or prevent the accumulation of A1AT polymers.
Using the example of A1AT deficiency as a representative IMD, this chapter has demonstrated the suitability of patient-specific hIPSC to model metabolic disorders affecting the liver. Indeed, hIPSCs derived from patient with specific IMDs can be easily differentiated into somatic cell types in which the disease manifests to address questions pertinent to the disease mechanism, the variability in disease phenotypes, and the development of treatment regimens. Thus, hIPSC-based hepatocyte platforms represent a physiologically relevant system to gain new understandings of IMDs and design strategies to reduce the burden of these diseases.
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