Translational Approaches for Human Liver Disease
Tuesday, February 20, 2018, 9:00 AM - 5:00 PM
The New York Academy of Sciences, 7 World Trade Center, 250 Greenwich St Fl 40, New York
The New York Academy of Sciences
Non-alcoholic steatohepatitis (NASH) is the second leading cause of disease driving the need for liver transplantation in the United States with no approved treatments currently available. While a number of clinical approaches are under investigation, historically many promising drug candidates have not demonstrated clinical benefit, in part due to an over-reliance on animal models.
In vitro approaches that incorporate primary human cell types to model disease have the potential to improve clinical success by bridging the gap from preclinical animal models to patients. This symposium will present cutting-edge research highlighting current and emerging in vitro model systems, covering potential benefits and limitations for the treatment of liver disease.
Scientific Organizing Committee
February 20, 2018
Registration and Continental Breakfast
Introduction and Welcome Remarks
Session 1: In Vitro Models of Liver Injury
Limitations of NASH Animal Models
A High-Throughput, Microphysiological System to recapitulate Liver Microenvironment, Function and Disease
Networking Coffee Break
An IPSC Model to Study Mechanisms of NAFLD Associated with Polymorphisms of PNPLA3
Non-alcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease in the adult and pediatric population. Genome-wide association studies (GWAS) have identified a polymorphism in the gene PNPLA3 that has a strong association with risk and severity of NAFLD, with the variant allele of PNPLA3 being associated with more severe biochemical and histological abnormalities. Although it is known that the protein product of PNPLA3 is involved in lipid metabolism, the exact function in humans is still unclear. Using TAL effector nuclease (TALEN) technology, TALENs specific to the PNLA3 SNP were designed to generate isogenic lines from human induced pluripotent cells (iPSC) with a known genetic background with the variant (Var) and wildtype (WT) homozygous alleles of PNPLA3. WT and Var iPSCs grow well in culture and readily differentiate to HLCs. We found that expression of PNPLA3 is decreased in Var HLCs. Interestingly, expression of other genes involved in lipolysis such as PNPLA2 and PPAR alpha is upregulated, which may suggest an adaptive mechanism to decreased function of PNPLA3. To test the hypothesis that the Var PNPLA3 allele confer its risk due to aberrant lipid metabolism resulting in lipotoxicity in the early onset of NAFLD, we compared intracellular lipid accumulation by Nile Red triglyceride (TG) staining. Using immunofluorescence and flow cytometry we found that PNPLA3 Var HLCs accumulate lipid droplets at baseline and upon exposure to palmitic acid (PA). Var HLCs upregulate autophagy as shown by increased levels of activated LC3, which may be a consequence of lipid accumulation as autophagy has been proposed as a mechanism of lipid turnover in hepatic cells. To further explore the link between lipid accumulation and inflammation, we used qPCR and a highly sensitive ELISA assay to measure expression and secretion of cytokines that are relevant to the development of inflammation in NAFLD. We found that PNPLA3 Var HLCs have increased expression of IL1b at baseline, and increased secretion of IL1a and IL6 upon PA treatment. In summary, we have generated and validated the first known human model carrying polymorphisms specific to the PNPLA3 locus to study mechanisms of NAFLD. Var HLCs have a strong phenotype of lipid accumulation that can be used as a suitable readout for high-throughput drug screening. We are currently testing compounds that ameliorate lipid accumulation and inflammatory phenotypes in PNPLA3 Var HLCs with a focus on PPAR alpha agonists and autophagy inducers. Our work will open the door to a new range of experimentation in elucidating the mechanism underlying the association between PNPLA3 and NAFLD, predictive diagnostics and therapeutic discovery.
Modeling Human Liver Disease Using Stem Cell Derived and Primary Human Hepatocyte Systems
Session 2: Data Blitz Talks
Identification of TAZ as a Therapeutic Target for Nonalcoholic Steatohepatitis
Preemptive activation of the integrated stress response protects mice from diet-induced obesity and insulin resistance via FGF21 induction
Regulation of Hepatocyte Cell Death by O-GlcNAc Transferase
Inhibition of the Prostaglandin Transporter attenuates Diet Induced Obesity and Liver Steatosis
Session 3: NASH and Tissue Models
Utility of an in vitro Human Liver System for Drug Discovery in Nonalcoholic Steatohepatitis and Liver Rare Diseases
Non-alcoholic fatty liver disease (NAFLD) is a rapidly emerging public health crisis, affecting up to 1/3 of the U.S. population and can progress to non-alcoholic steatohepatitis (NASH) and cirrhosis, often resulting in liver transplant or death. This is one of the most active areas in drug discovery with over 40 drugs in the preclinical and clinical phase of development, but none-to-date advancing beyond Phase III clinical trials or approved for therapy. The field is hampered by a lack of disease understanding and rodent models that do not reliably map to the human response. In this presentation, we describe an in vitro approach that combines primary human hepatocytes, stellate cells, and macrophages, a physiologically relevant tissue microenvironment consisting of liver sinusoid hemodynamic and transport conditions, and clinically-derived concentrations of NASH risk factors to mimic aspects of human NASH in vitro. This system captures critical pathophysiological drivers of NASH such as steatosis, inflammation, and fibrosis and has been validated against human NASH biopsy samples (Feaver et al, JCI Insight, 2017) and with leading clinical stage drugs. We have utilized the in vitro human NASH model to survey the current drug-development landscape in order to understand the therapeutic gaps, providing the principal dataset to identity new targets for NASH therapies. This concept is also being applied to identify novel targets and develop new therapies to treat pediatric rare liver diseases (Chapman et al, Mol. Genet. Metab. 2016) and in the future, rare vascular diseases (Collado et al, Stem Cells Transl Med 2017).
Microphysiological Systems with Liver Module
Effective human surrogates constructed from a combination of human tissue engineered constructs, microfabricated devices, and PBPK (physiologically based pharmacokinetic) models offer a potential alternative or supplement to animal studies to make better decisions on which drug candidates to move into clinical trials. I will focus on multi-organ microphysiological systems, which are also known as “Body-on-a-Chip” systems. We have constructed such systems with 2 to 13 “organ” compartments using a self-contained “pumpless” system that is low cost and robust in operation. A key organ is the liver as it is the primary site of drug metabolism. A multi-organ system allows investigation in a preclinical model of human response to drugs and its metabolites some of which may cause organ toxicity. In addition to measuring metabolic responses, we measure electrical activity and force generation of various cell types in such devices while using a serum free medium (in collaboration with J. Hickman, University of Central Florida). These studies provide direct information on drug effects on particular organs. With electrically active cells, responses can be measured within minutes of addition of a drug. Such systems can be used to address issues of efficacy and toxicity in preclinical studies leading to better choices of which drugs to take into clinical trials. It is possible to model not only healthy tissue but to examine the potential response if an organ is diseased. We believe that preclinical evaluation of a drug in a multi-organ human model will be of significant benefit.
Networking Coffee Break
Liver Chip for Studying Human Liver Function and Disease Development
Modeling Liver Fibrosis In Vitro: Bridging the Gap Using a 3D Approach
Liver fibrosis poses an important human health concern from both a clinical and regulatory standpoint. Because fibrosis develops over time from a sequence of complex and cumulative interactions between hepatocytes and non-parenchymal cells, it has proven challenging to model using standard in vitro and preclinical in vivo models. To better understand the series of initiating and early adaptive events that occur among resident hepatic cells to mediate or remediate this abnormal wound healing response, we took a three-dimensional approach using a commercially available bioprinted liver model comprising primary human hepatocytes, human umbilical vein endothelial cells, and hepatic stellate cells. Because these cultures sustain important cell-cell interactions and liver specific functions over an extended period of time, we assessed the utility of this novel platform to model fundamental aspects of fibrotic liver injury by performing a repeated low concentration exposure regimen to prototype fibrogenic agents to precipitate a fibrogenic response. Here we present compelling evidence of compound-induced fibrogenesis following extended compound exposure and highlight the utility of the model in assessing the progression of complex, multicellular toxicity responses and disease processes such as non-alcoholic steatohepatitis (NASH). We further demonstrate bioprinted human liver tissues are well-suited to model and examine temporal fibrogenic events in vitro and their utility in beginning to understand the early events underlying fibrotic injury. Overall, this work lays the foundation for a well-defined, dynamic model of compound-induced liver fibrosis progression and regression with additional implications in screening antifibrotic drug efficacy and liability during preclinical development.
Characterizing the Role of PRMT5 in Liver Regeneration and Carcinogenesis
Arginine methylation is a major post-translational modification that regulates a myriad of biological processes including transcription, pre-mRNA splicing, mRNA translation and cell signalling. PRMT5 is the major mammalian type II arginine methyltransferase and plays an essential role in regulating gene expression and pre-mRNA splicing. Constitutive genetic ablation of PRMT5 causes early embryonic lethality (E3.5) and maintenance of its activity in proliferating cells is required for growth and survival. Therefore, it remains unclear if and how PRMT5 plays a role during hepatogenesis and in the adult liver during liver regeneration.
In our current studies, we are investigating the role of PRMT5 during liver organogenesis, during partial hepatectomy and in liver cancer.
Our preliminary data link PRMT5 with the regulation of the p53 and WNT pathway, which are both important in normal physiological and pathological conditions. Given the current testing pf PRMT5 inhibitors in clinical trials we highlight potential risk of liver toxicity and benefits to target PRMT5 in liver cancer.
Panel Discussion: Working Towards a Predictive in vitro NASH Model