Dagmara Szkolnicka

Developing High-Fidelity Hepatotoxicity Models From Pluripotent Stem Cells

Faithfully recapitulating human physiology “in a dish” from a renewable source remains a holy grail for medicine and pharma. Many procedures have been described that, to a limited extent, exhibit human tissue‐specific function in vitro. In particular, incomplete cellular differentiation and/or the loss of cell phenotype postdifferentiation play a major part in this void. We have developed an interdisciplinary approach to address this problem, using skill sets in cell biology, materials chemistry, and pharmacology. Pluripotent stem cells were differentiated to hepatocytes before being replated onto a synthetic surface. Our approach yielded metabolically active hepatocyte populations that displayed stable function for more than 2 weeks in vitro. Although metabolic activity was an important indication of cell utility, the accurate prediction of cellular toxicity in response to specific pharmacological compounds represented our goal. Therefore, detailed analysis of hepatocellular toxicity was performed in response to a custom‐built and well‐defined compound set and compared with primary human hepatocytes. Importantly, stem cell‐derived hepatocytes displayed equivalence to primary human material. Moreover, we demonstrated that our approach was capable of modeling metabolic differences observed in the population. In conclusion, we report that pluripotent stem cell‐derived hepatocytes will model toxicity predictably and in a manner comparable to current gold standard assays, representing a major advance in the field.

Gene networks and transcription factor motifs defining the differentiation of stem cells into hepatocyte-like cells

Graphical abstract

Accurate Prediction of Drug-Induced Liver Injury Using Stem Cell-Derived Populations

Despite major progress in the knowledge and management of human liver injury, there are millions of people suffering from chronic liver disease. Currently, the only cure for end‐stage liver disease is orthotopic liver transplantation; however, this approach is severely limited by organ donation. Alternative approaches to restoring liver function have therefore been pursued, including the use of somatic and stem cell populations. Although such approaches are essential in developing scalable treatments, there is also an imperative to develop predictive human systems that more effectively study and/or prevent the onset of liver disease and decompensated organ function. We used a renewable human stem cell resource, from defined genetic backgrounds, and drove them through developmental intermediates to yield highly active, drug‐inducible, and predictive human hepatocyte populations. Most importantly, stem cell‐derived hepatocytes displayed equivalence to primary adult hepatocytes, following incubation with known hepatotoxins. In summary, we have developed a serum‐free, scalable, and shippable cell‐based model that faithfully predicts the potential for human liver injury. Such a resource has direct application in human modeling and, in the future, could play an important role in developing renewable cell‐based therapies.

Recombinant Laminins Drive the Differentiation and Self-Organization of hESC-Derived Hepatocytes

Summary Stem cell-derived somatic cells represent an unlimited resource for basic and translational science. Although promising, there are significant hurdles that must be overcome. Our focus is on the generation of the major cell type of the human liver, the hepatocyte. Current protocols produce variable populations of hepatocytes that are the product of using undefined components in the differentiation process. This serves as a significant barrier to scale-up and application. To tackle this issue, we designed a defined differentiation process using recombinant laminin substrates to provide instruction. We demonstrate efficient hepatocyte specification, cell organization, and significant improvements in cell function and phenotype. This is driven in part by the suppression of unfavorable gene regulatory networks that control cell proliferation and migration, pluripotent stem cell self-renewal, and fibroblast and colon specification. We believe that this represents a significant advance, moving stem cell-based hepatocytes closer toward biomedical application.

Pluripotent Stem Cell–Derived Hepatocytes: Potential and Challenges in Pharmacology

The liver is a fascinating organ and performs a wide range of functions necessary for life. Because the hepatocyte is the major functional cell type found in the liver, it is important that we better understand its role in health and disease. Functional hepatocytes have been derived from many sources, including human stem cell populations. These models offer new opportunities to further our understanding of human liver biology from diverse genotypes and, in the future, to facilitate the development of novel medicines or cell-based therapies. This review discusses limitations in current cell-based models and the advantages offered by pluripotent stem cell-derived hepatocytes.

Modulating Innate Immunity Improves Hepatitis C Virus Infection and Replication in Stem Cell-Derived Hepatocytes

Summary In this study, human embryonic stem cell-derived hepatocytes (hESC-Heps) were investigated for their ability to support hepatitis C virus (HCV) infection and replication. hESC-Heps were capable of supporting the full viral life cycle, including the release of infectious virions. Although supportive, hESC-Hep viral infection levels were not as great as those observed in Huh7 cells. We reasoned that innate immune responses in hESC-Heps may lead to the low level of infection and replication. Upon further investigation, we identified a strong type III interferon response in hESC-Heps that was triggered by HCV. Interestingly, specific inhibition of the JAK/STAT signaling pathway led to an increase in HCV infection and replication in hESC-Heps. Of note, the interferon response was not evident in Huh7 cells. In summary, we have established a robust cell-based system that allows the in-depth study of virus-host interactions in vitro.

Fluid shear stress modulation of hepatocyte-like cell function

Freshly isolated human adult hepatocytes are considered to be the gold standard tool for in vitro studies. However, primary hepatocyte scarcity, cell cycle arrest and the rapid loss of cell phenotype limit their widespread deployment. Human embryonic stem cells and induced pluripotent stem cells provide renewable sources of hepatocyte-like cells (HLCs). Despite the use of various differentiation methodologies, HLCs like primary human hepatocytes exhibit unstable phenotype in culture. It has been shown that the functional capacity can be improved by adding back elements of human physiology, such as cell co-culture or through the use of natural and/or synthetic surfaces. In this study, the effect of fluid shear stress on HLC performance was investigated. We studied two important liver functions, cytochrome P450 drug metabolism and serum protein secretion, in static cultures and those exposed to fluid shear stress. Our study demonstrates that fluid shear stress improved Cyp1A2 activity by approximately fivefold. This was paralleled by an approximate ninefold increase in sensitivity to a drug, primarily metabolised by Cyp2D6. In addition to metabolic capacity, fluid shear stress also improved hepatocyte phenotype with an approximate fourfold reduction in the secretion of a foetal marker, alpha-fetoprotein. We believe these studies highlight the importance of introducing physiologic cues in cell-based models to improve somatic cell phenotype.

Reducing Hepatocyte Injury and Necrosis in Response to Paracetamol Using Noncoding RNAs

The liver performs multiple functions within the human body. It is composed of numerous cell types, which play important roles in organ physiology. Our study centers on the major metabolic cell type of the liver, the hepatocyte, and its susceptibility to damage during drug overdose. In these studies, hepatocytes were generated from a renewable and genetically defined resource. In vitro‐derived hepatocytes were extensively profiled and exposed to varying levels of paracetamol and plasma isolated from liver‐failure patients, with a view to identifying noncoding microRNAs that could reduce drug‐ or serum‐induced hepatotoxicity. We identified a novel anti‐microRNA, which reduced paracetamol‐induced hepatotoxicity and glutathione depletion. Additionally, we identified a prosurvival role for anti‐microRNA‐324 following exposure to plasma collected from liver failure patients. We believe that these studies represent an important advance for the field, demonstrating the power of stem cell‐derived systems to model human biology “in a dish” and identify novel noncoding microRNAs, which could be translated to the clinic in the future.

Polymer Supported Directed Differentiation Reveals a Unique Gene Signature Predicting Stable Hepatocyte Performance

In theory, pluripotent stem cells can give rise to all somatic cell types found in the human body. The ability to generate renewable sources of human cells has enormous potential to improve human health and wealth. One major obstacle to the routine deployment of stem cell-derived cells is their instability in culture. To tackle this issue a synthetic polymer surface is used.

Concise Review: Advances in Generating Hepatocytes from Pluripotent Stem Cells for Translational Medicine

The liver is one of the major organs in the human body. Severe or prolonged exposure of the liver to different factors may cause life‐threatening disease, which necessitates donor organ transplantation. While orthotopic liver transplantation can be used to effectively treat liver failure, it is an invasive procedure, which is severely limited by organ donation. Therefore, alternative sources of liver support have been proposed and studied. This includes the use of pluripotent stem cell‐derived hepatocytes as a renewable source of cells for therapy. In addition to cell‐based therapies, in vitro engineered liver tissue provides powerful models for human drug discovery and disease modeling. This review focuses on the generation of hepatocyte‐like cells from pluripotent stem cells and their application in translational medicine. Stem Cells 2016;34:1421–1426