A human iPSC-derived hepatocyte screen identifies compounds that inhibit production of Apolipoprotein B.

Familial hypercholesterolemia (FH) patients suffer from excessively high levels of Low Density Lipoprotein Cholesterol (LDL-C), which can cause severe cardiovascular disease. Statins, bile acid sequestrants, PCSK9 inhibitors, and cholesterol absorption inhibitors are all inefficient at treating FH patients with homozygous LDLR gene mutations (hoFH). Drugs approved for hoFH treatment control lipoprotein production by regulating steady-state Apolipoprotein B (apoB) levels. Unfortunately, these drugs have side effects including accumulation of liver triglycerides, hepatic steatosis, and elevated liver enzyme levels. To identify safer compounds, we used an iPSC-derived hepatocyte platform to screen a structurally representative set of 10,000 small molecules from a proprietary library of 130,000 compounds. The screen revealed molecules that could reduce the secretion of apoB from cultured hepatocytes and from humanized livers in mice. These small molecules are highly effective, do not cause abnormal lipid accumulation, and share a chemical structure that is distinct from any known cholesterol lowering drug.

FXR inhibition may protect from SARS-CoV-2 infection by reducing ACE2.

Preventing SARS-CoV-2 infection by modulating viral host receptors, such as angiotensin-converting enzyme 2 (ACE2)1, could represent a new chemoprophylactic approach for COVID-19 that complements vaccination2,3. However, the mechanisms that control the expression of ACE2 remain unclear. Here we show that the farnesoid X receptor (FXR) is a direct regulator of ACE2 transcription in several tissues affected by COVID-19, including the gastrointestinal and respiratory systems. We then use the over-the-counter compound z-guggulsterone and the off-patent drug ursodeoxycholic acid (UDCA) to reduce FXR signalling and downregulate ACE2 in human lung, cholangiocyte and intestinal organoids and in the corresponding tissues in mice and hamsters. We show that the UDCA-mediated downregulation of ACE2 reduces susceptibility to SARS-CoV-2 infection in vitro, in vivo and in human lungs and livers perfused ex situ. Furthermore, we reveal that UDCA reduces the expression of ACE2 in the nasal epithelium in humans. Finally, we identify a correlation between UDCA treatment and positive clinical outcomes after SARS-CoV-2 infection using retrospective registry data, and confirm these findings in an independent validation cohort of recipients of liver transplants. In conclusion, we show that FXR has a role in controlling ACE2 expression and provide evidence that modulation of this pathway could be beneficial for reducing SARS-CoV-2 infection, paving the way for future clinical trials.

Chromatin remodeling is restricted by transient GATA6 binding during iPSC differentiation to definitive endoderm.

In addition to cooperatively driving transcriptional programs, emerging evidence supports transcription factors interacting with one another to modulate the outcome of binding events. As such, transcription factor interactions fine-tune the unique gene expression profiles required for developmental progression. Using human-induced pluripotent stem cells as a model of human endoderm lineage commitment, we reveal that GATA6 transiently co-localizes with EOMES at regions associated with non-endodermal lineages and is required for the repression of chromatin opening at these loci. Our results indicate that GATA6-dependent repression of chromatin remodeling, which is potentially mediated via the recruitment of NCOR1 to the EOMES interactome, contributes to definitive endoderm commitment. We anticipate that similar mechanisms are common during human development, furthering our understanding of the complex mechanisms that define cell fate decisions.

GATA6 defines endoderm fate by controlling chromatin accessibility during differentiation of human-induced pluripotent stem cells.

In addition to driving specific gene expression profiles, transcriptional regulators are becoming increasingly recognized for their capacity to modulate chromatin structure. GATA6 is essential for the formation of definitive endoderm; however, the molecular basis defining the importance of GATA6 to endoderm commitment is poorly understood. The members of the GATA family of transcription factors have the capacity to bind and alter the accessibility of chromatin. Using pluripotent stem cells as a model of human development, we reveal that GATA6 is integral to the establishment of the endoderm enhancer network via the induction of chromatin accessibility and histone modifications. We additionally identify the chromatin-modifying complexes that interact with GATA6, defining the putative mechanisms by which GATA6 modulates chromatin architecture. The identified GATA6-dependent processes further our knowledge of the molecular mechanisms that underpin cell-fate decisions during formative development.

PDE5 inhibition rescues mitochondrial dysfunction and angiogenic responses induced by Akt3 inhibition by promotion of PRC expression.

Akt3 regulates mitochondrial content in endothelial cells through the inhibition of PGC-1α nuclear localization and is also required for angiogenesis. However, whether there is a direct link between mitochondrial function and angiogenesis is unknown. Here we show that Akt3 depletion in primary endothelial cells results in decreased uncoupled oxygen consumption, increased fission, decreased membrane potential, and increased expression of the mitochondria-specific protein chaperones, HSP60 and HSP10, suggesting that Akt3 is required for mitochondrial homeostasis. Direct inhibition of mitochondrial homeostasis by the model oxidant paraquat results in decreased angiogenesis, showing a direct link between angiogenesis and mitochondrial function. Next, in exploring functional links to PGC-1α, the master regulator of mitochondrial biogenesis, we searched for compounds that induce this process. We found that, sildenafil, a phosphodiesterase 5 inhibitor, induced mitochondrial biogenesis as measured by increased uncoupled oxygen consumption, mitochondrial DNA content, and voltage-dependent anion channel protein expression. Sildenafil rescued the effects on mitochondria by Akt3 depletion or pharmacological inhibition and promoted angiogenesis, further supporting that mitochondrial homeostasis is required for angiogenesis. Sildenafil also induces the expression of PGC-1 family member PRC and can compensate for PGC-1α activity during mitochondrial stress by an Akt3-independent mechanism. The induction of PRC by sildenafil depends upon cAMP and the transcription factor CREB. Thus, PRC can functionally substitute during Akt3 depletion for absent PGC-1α activity to restore mitochondrial homeostasis and promote angiogenesis. These findings show that mitochondrial homeostasis as controlled by the PGC family of transcriptional activators is required for angiogenic responses.

FoxA factors: the chromatin key and doorstop essential for liver development and function.

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.

Enhanced genome editing in human iPSCs with CRISPR-CAS9 by co-targeting ATP1a1.

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-targeted ATP1A1 and 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-targeting ATP1A1 with the second locus enriches for mutagenesis events through cardiac glycoside selection in human iPSCs.

The Use of Human Pluripotent Stem Cells for Modeling Liver Development and Disease.

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.

Donor-Dependent and Other Nondefined Factors Have Greater Influence on the Hepatic Phenotype Than the Starting Cell Type in Induced Pluripotent Stem Cell Derived Hepatocyte-Like Cells.

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.

Mechanistic evaluation of primary human hepatocyte culture using global proteomic analysis reveals a selective dedifferentiation profile.

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 <1 %, of which 1117 were quantified at every time point. Increasing numbers of significantly differentially expressed proteins compared with the freshly isolated cells were observed at 24 h (40 proteins), 72 h (118 proteins) and 168 h (272 proteins) (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.

Concise review: workshop review: understanding and assessing the risks of stem cell-based therapies.

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.

MicroRNA-122: a novel hepatocyte-enriched in vitro marker of drug-induced cellular toxicity.

Emerging hepatic models for the study of drug-induced toxicity include pluripotent stem cell-derived hepatocyte-like cells (HLCs) and complex hepatocyte-non-parenchymal cellular coculture to mimic the complex multicellular interactions that recapitulate the niche environment in the human liver. However, a specific marker of hepatocyte perturbation, required to discriminate hepatocyte damage from non-specific cellular toxicity contributed by non-hepatocyte cell types or immature differentiated cells is currently lacking, as the cytotoxicity assays routinely used in in vitro toxicology research depend on intracellular molecules which are ubiquitously present in all eukaryotic cell types. In this study, we demonstrate that microRNA-122 (miR-122) detection in cell culture media can be used as a hepatocyte-enriched in vitro marker of drug-induced toxicity in homogeneous cultures of hepatic cells, and a cell-specific marker of toxicity of hepatic cells in heterogeneous cultures such as HLCs generated from various differentiation protocols and pluripotent stem cell lines, where conventional cytotoxicity assays using generic cellular markers may not be appropriate. We show that the sensitivity of the miR-122 cytotoxicity assay is similar to conventional assays that measure lactate dehydrogenase activity and intracellular adenosine triphosphate when applied in hepatic models with high levels of intracellular miR-122, and can be multiplexed with other assays. MiR-122 as a biomarker also has the potential to bridge results in in vitro experiments to in vivo animal models and human samples using the same assay, and to link findings from clinical studies in determining the relevance of in vitro models being developed for the study of drug-induced liver injury.