James A. Heslop

Glycogen storage disease 1A (GSD1A) is an inherited metabolic disorder caused by glucose-6-phosphatase (G6PC) deficiency. Patients with GSD1A present disturbed glucose homeostasis and exhibit glycogen accumulation accompanied by hepatomegaly, hypoglycemia, lactic acidosis and hyperlipidemia. However, there are currently no licensed treatments for GSD1A, and human in vitro systems for disease modelling and drug screening are lacking. We aimed to develop a human hepatocyte model recapitulating the GSD1A phenotype in-a-dish. Healthy and GSD1A patient-derived (R83C) induced pluripotent stem cells (iPSCs), alongside isogenic controls, were differentiated towards hepatocyte-like cells (HLCs). Successful differentiation was confirmed by expression of the hepatocyte markers Albumin, Alpha-1-antitrypsin, Alpha-fetoprotein and HNF4alpha using qPCR and immunocytochemistry. Mutation correction (C83R) in G6PC was performed by CRISPR/Cas9 mediated gene editing, and iPSC genotyping was confirmed by Sanger sequencing. Media glucose secretion and intracellular glycogen levels were measured by colorimetric assays. Intracellular glycogen levels were significantly reduced in healthy HLCs following 1h glucagon stimulation under starvation. However, no differences in GSD1A-derived HLCs were observed. In line with this, media glucose levels were elevated in healthy glucagon-stimulated HLCs compared to vehicle-treated ones, but not in GSD1A-HLCs, suggesting impaired glycogen breakdown towards glucose formation. In contrast, correction of disease mutation in GSD1A-HLCs reduced glycogen levels in a time dependent manner, alongside enhanced glucagon-stimulated glycogen breakdown. Finally, to confirm that the observed effects were the result of corrected G6Pase activity, transient over-expression of G6PC in unedited GSD1A-HLCs similarly resulted in increased glucose secretion, suggestive of restored glycogen mobilization. We have developed an iPSC-derived hepatocyte model that recapitulates human GSD1A phenotype in vitro. This technology provides a framework for the development of human liver disease models from patients of varied genetic disease backgrounds. Crucially, it highlights the advantage of iPSCs as an effective platform for liver disease modelling and hit-lead drug screening.

Pioneer factors are transcriptional regulators with the capacity to bind inactive regions of chromatin and induce changes in accessibility that underpin cell fate decisions. The FOXA family of transcription factors is well understood to have pioneer capacity. Indeed, researchers have uncovered numerous examples of FOXA-dependent epigenomic modulation in developmental and disease processes. Despite the presence of FOXA being essential for correct epigenetic patterning, the need for continued FOXA presence postchromatin modulation has been debated. In a recent study in this issue of Genes & Development, Reizel and colleagues (pp. 1039–1050) show that the tissue-specific ablation of FOXA1/2/3 in the adult mouse liver results in the collapse of the epigenetic profile that maintains the hepatic gene expression profile. Thus, FOXA functions as a key, opening regions of chromatin during development, and as a doorstep, maintaining the established euchromatic structure in adult tissue.

Genome editing in human induced pluripotent stem cells (iPSCs) provides the potential for disease modeling and cell therapy. By generating iPSCs with specific mutations, researchers can differentiate the modified cells to their lineage of interest for further investigation. However, the low efficiency of targeting in iPSCs has hampered the application of genome editing. In this study we used a CRISPR-Cas9 system that introduces a specific point substitution into the sequence of the Na⁺/K⁺-ATPase subunit ATP1A1. The introduced mutation confers resistance to cardiac glycosides, which can then be used to select successfully targeted cells. Using this system, we introduced different formats of donor DNA for homology-directed repair (HDR), including single-strand DNAs, double-strand DNAs, and plasmid donors. We achieved a 35-fold increase in HDR when using plasmid donor with a 400 bp repair template. We further co-targetedATP1A1and a second locus of interest to determine the enrichment of mutagenesis after cardiac glycoside selection. Through this approach, INDEL rate was increased after cardiac glycoside treatment, while HDR enrichment was only observed at certain loci. Collectively, these results suggest that a plasmid donor with a 400 bp repair template is an optimal donor DNA for targeted substitution and co-targetingATP1A1with the second locus enriches for mutagenesis events through cardiac glycoside selection in human iPSCs.

The use of pluripotent stem cells (PSCs) has transformed the investigation of liver development and disease. Clinical observations and animal models have provided the foundations of our understanding in these fields. While animal models remain essential research tools, long experimental lead times and low throughput limit the scope of investigations. The ability of PSCs to produce large numbers of human hepatocyte‐like cells, with a given or modified genetic background, allows investigators to use previously incompatible experimental techniques, such as high‐throughput screens, to enhance our understanding of liver development and disease. In this review, we explore how PSCs have expedited our understanding of developmental mechanisms and have been used to identify new therapeutic options for numerous hepatic diseases. We discuss the future directions of the field, including how to further unlock the potential of the PSC model to make it amenable for use with a broader range of assays and a greater repertoire of diseases. Furthermore, we evaluate the current weaknesses of the PSC model and the directions open to researchers to address these limitations. Conclusion: The use of PSCs to model human liver disease and development has and will continue to have substantial impact, which is likely to further expand as protocols used to generate hepatic cells are improved.

Drug-induced liver injury is the greatest cause of post-marketing drug withdrawal; therefore, substantial resources are directed toward triaging potentially dangerous new compounds at all stages of drug development. One of the major factors preventing effective screening of new compounds is the lack of a predictive in vitro model of hepatotoxicity. Primary human hepatocytes offer a metabolically relevant model for which the molecular initiating events of hepatotoxicity can be examined; however, these cells vary greatly between donors and dedifferentiate rapidly in culture. Induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) offer a reproducible, physiologically relevant and genotypically normal model cell; however, current differentiation protocols produce HLCs with a relatively immature phenotype. During the reprogramming of somatic cells, the epigenome undergoes dramatic changes; however, this “resetting” is a gradual process, resulting in an altered differentiation propensity, skewed toward the lineage of origin, particularly in early passage cultures. We, therefore, performed a comparison of human hepatocyte- and dermal fibroblast-derived iPSCs, assessing the impact of epigenetic memory at all stages of HLC differentiation. These results provide the first isogenic assessment of the starting cell type in human iPSC-derived HLCs. Despite a trend toward improvement in hepatic phenotype in albumin secretion and gene expression, few significant differences in hepatic differentiation capacity were found between hepatocyte and fibroblast-derived iPSCs. We conclude that the donor and inter-clonal differences have a greater influence on the hepatocyte phenotypic maturity than the starting cell type. Therefore, it is not necessary to use human hepatocytes for generating iPSC-derived HLCs. Stem Cells Translational Medicine 2017;6:1321–1331

The application of primary human hepatocytes following isolation from human tissue is well accepted to be compromised by the process of dedifferentiation. This phenomenon reduces many unique hepatocyte functions, limiting their use in drug disposition and toxicity assessment. The aetiology of dedifferentiation has not been well defined, and further understanding of the process would allow the development of novel strategies for sustaining the hepatocyte phenotype in culture or for improving protocols for maturation of hepatocytes generated from stem cells. We have therefore carried out the first proteomic comparison of primary human hepatocyte differentiation. Cells were cultured for 0, 24, 72 and 168 h as a monolayer in order to permit unrestricted hepatocyte dedifferentiation, so as to reveal the causative signalling pathways and factors in this process, by pathway analysis. A total of 3430 proteins were identified with a false detection rate of p < 0.05). In particular, cytochromes P450 and mitochondrial proteins underwent major changes, confirmed by functional studies and investigated by pathway analysis. We report the key factors and pathways which underlie the loss of hepatic phenotype in vitro, particularly those driving the large-scale and selective remodelling of the mitochondrial and metabolic proteomes. In summary, these findings expand the current understanding of dedifferentiation should facilitate further development of simple and complex hepatic culture systems.

It is evident from the drug development failures of the past 30 years that only through a better mechanistic understanding of specific drug-induced liver injury (DILI) will we improve our ability to predict clinical hepatotoxicity. Current in vitro models used for the prediction of DILI often suffer from uninformative, inappropriate, and poorly translatable endpoint measurements, and this may lead to unreliable inferences being drawn. Moreover, there is currently a significant disconnect between in vitro DILI endpoints and those used to assess hepatotoxicity in a clinical setting. This perspective article aims to identify the gaps in this area and reviews up-to-date information on current approaches to apply a human context to laboratory in vitro models. In order to develop better models to predict more accurately which chemicals will elicit hepatotoxicity in humans, a coordinated research effort is required involving the pharmaceutical industry, academia, and regulatory bodies. Key to this is selection and prioritization of endpoints of hepatocellular perturbation, as well as the definition of appropriate biomarkers, that truly reflect events occurring in the liver in vivo. We suggest that in order to improve our ability to explore mechanisms implicated in DILI, and thereby enhance the predictive power of in vitro test systems, there is an absolute need for better access to patient samples for use in exploratory biomarker development that better reflect specific mechanisms of hepatic injury, particularly those from industry-sponsored prospective clinical trials, and for improved phenotypic characterization of patients susceptible to DILI.

The field of stem cell therapeutics is moving ever closer to widespread application in the clinic. However, despite the undoubted potential held by these therapies, the balance between risk and benefit remains difficult to predict. As in any new field, a lack of previous application in man and gaps in the underlying science mean that regulators and investigators continue to look for a balance between minimizing potential risk and ensuring therapies are not needlessly kept from patients. Here, we attempt to identify the important safety issues, assessing the current advances in scientific knowledge and how they may translate to clinical therapeutic strategies in the identification and management of these risks. We also investigate the tools and techniques currently available to researchers during preclinical and clinical development of stem cell products, their utility and limitations, and how these tools may be strategically used in the development of these therapies. We conclude that ensuring safety through cutting-edge science and robust assays, coupled with regular and open discussions between regulators and academic/industrial investigators, is likely to prove the most fruitful route to ensuring the safest possible development of new products.