Pluripotent stem cell-derived hepatocyte-like cells

Liver disease is an important clinical problem, impacting over 30 million Americans and over 600 million people worldwide. It is the 12th leading cause of death in the United States and the 16th worldwide. Due to a paucity of donor organs, several thousand Americans die yearly while waiting for liver transplantation. Unfortunately, alternative tissue sources such as fetal hepatocytes and hepatic cell lines are unreliable, difficult to reproduce, and do not fully recapitulate hepatocyte phenotype and functions. As a consequence, alternative cell sources that do not have these limitations have been sought. Human embryonic stem (hES) cell- and induced pluripotent stem (iPS) cell-derived hepatocyte-like cells may enable cell based therapeutics, the study of the mechanisms of human disease and human development, and provide a platform for screening the efficacy and toxicity of pharmaceuticals. iPS cells can be differentiated in a step-wise fashion with high efficiency and reproducibility into hepatocyte-like cells that exhibit morphologic and phenotypic characteristics of hepatocytes. In addition, iPS-derived hepatocyte-like cells (iHLCs) possess some functional hepatic activity as they secrete urea, alpha-1-antitrypsin, and albumin. However, the combined phenotypic and functional traits exhibited by iHLCs resemble a relatively immature hepatic phenotype that more closely resembles that of fetal hepatocytes rather than adult hepatocytes. Specifically, iHLCs express fetal markers such as alpha-fetoprotein and lack key mature hepatocyte functions, as reflected by drastically reduced activity (~ 0.1%) of important detoxification enzymes (i.e. CYP2A6, CYP3A4). These key differences between iHLCs and primary adult human hepatocytes have limited the use of stem cells as a renewable source of functional adult hepatocytes for in vitro and in vivo applications. Unfortunately, the developmental pathways that control hepatocyte maturation from a fetal into an adult hepatocyte are poorly understood, which has hampered the field in its efforts to induce further maturation of iPS-derived hepatic lineage cells. This review analyzes recent developments in the derivation of hepatocyte-like cells, and proposes important points to consider and assays to perform during their characterization. In the future, we envision that iHLCs will be used as in vitro models of human disease, and in the longer term, provide an alternative cell source for drug testing and clinical therapy.National Institutes of Health (U.S.) (Roadmap for Medical Research Grant 1 R01 DK085713-01))American Gastroenterological Association (Research Scholar Award

Quantum dots to monitor RNAi delivery and improve gene silencing

A critical issue in using RNA interference for identifying genotype/phenotype correlations is the uniformity of gene silencing within a cell population. Variations in transfection efficiency, delivery-induced cytotoxicity and ‘off target’ effects at high siRNA concentrations can confound the interpretation of functional studies. To address this problem, we have developed a novel method of monitoring siRNA delivery that combines unmodified siRNA with seminconductor quantum dots (QDs) as multi color biological probes. We co-transfected siRNA with QDs using standard transfection techniques, thereby leveraging the photostable fluorescent nanoparticles to track delivery of nucleic acid, sort cells by degree of transfection and purify homogenously-silenced subpopulations. Compared to alternative RNAi tracking methods (co-delivery of reporter plasmids and end-labeling the siRNA), QDs exhibit superior photostability and tunable optical properties for an extensive selection of non-overlapping colors. Thus this simple, modular system can be extended toward multiplexed gene knockdown studies, as demonstrated in a two color proof-of-principle study with two biological targets. When the method was applied to investigate the functional role of T-cadherin (T-cad) in cell–cell communication, a subpopulation of highly silenced cells obtained by QD labeling was required to observe significant downstream effects of gene knockdown

Microenvironmental regulation of the sinusoidal endothelial cell phenotype in vitro

Author Manuscript: 2010 June 23.Liver sinusoidal endothelial cells (LSECs) differ, both structurally and functionally, from endothelial cells (ECs) lining blood vessels of other tissues. For example, in contrast to other ECs, LSECs possess fenestrations, have low detectable levels of platelet endothelial cell adhesion molecule 1 expression, and in rat tissue, they distinctively express a cell surface marker recognized by the SE-1 antibody. These unique phenotypic characteristics seen in hepatic tissue are lost over time upon culture in vitro; therefore, this study sought to systematically examine the effects of microenvironmental stimuli—namely, extracellular matrix and neighboring cells, on the LSEC phenotype in vitro. In probing the role of the underlying extracellular matrix, we identified collagen I and collagen III as well as mixtures of collagen I/collagen IV/fibronectin as having a positive effect on LSEC survival. Furthermore, using a stable hepatocellular model (hepatocyte–fibroblast) we were able to prolong the expression of both SE-1 and phenotypic functions of LSEC such as factor VIII activity and AcLOL uptake in cocultured LSECs through the production of short-range paracrine signals. In the course of these experiments, we identified the antigen recognized by SE-1 as CD32b. Conclusion: Collectively, this study has identified several microenvironmental regulators of liver sinusoidal endothelial cells that prolong their phenotypic functions for up to 2 weeks in culture, enabling the development of better in vitro models of liver physiology and disease

Real-time imaging of hepatitis C virus infection using a fluorescent cell-based reporter system

Author Manuscript 2010 August 1Hepatitis C virus (HCV), which infects 2–3% of the world population, is a causative agent of chronic hepatitis and the leading indication for liver transplantation1. The ability to propagate HCV in cell culture (HCVcc) is a relatively recent breakthrough and a key tool in the quest for specific antiviral therapeutics. Monitoring HCV infection in culture generally involves bulk population assays, use of genetically modified viruses and/or terminal processing of potentially precious samples. Here we develop a cell-based fluorescent reporter system that allows sensitive distinction of individual HCV-infected cells in live or fixed samples. We demonstrate use of this technology for several previously intractable applications, including live-cell imaging of viral propagation and host response, as well as visualizing infection of primary hepatocyte cultures. Integration of this reporter with modern image-based analysis methods could open new doors for HCV research.New York (State). Dept. of Health (Empire State Stem Cell Fund Contract C023046)United States. Public Health Service (Grant R01 DK56966)National Institutes of Health (U.S.) (Roadmap for Medical Research Grant 1 R01 DK085713-01)Howard Hughes Medical Institute (Investigator

Hepatitis C Virus Infection Induces Autocrine Interferon Signaling by Human Liver Endothelial Cells and Release of Exosomes, Which Inhibits Viral Replication

Liver sinusoidal endothelial cells (LSECs) make up a large proportion of the non-parenchymal cells in the liver. LSECs are involved in induction of immune tolerance, but little is known about their functions during hepatitis C virus (HCV) infection

Micropatterned co-cultures of hepatocytes and nonparenchymal cells : mechanisms of differentiation, dynamics and applications

Engineered liver tissue has the potential to support patients with hepatic dysfunction as well as provide an in vitro model of the liver for pharmaceutical drug development. Such applications mandate the use of primary hepatocytes- the parenchymal cell of the liver; however, hepatocytes rapidly lose viability and phenotypic functions in vitro. Co-cultivation of hepatocytes with nonparenchymal cells (NPC) has been reported to prevent this deterioration. Several aspects of this so-called 'co- culture effect' remain incompletely understood: the molecular mechanisms by which NPC stabilize liver-specific functions, dynamics of interaction (i.e. whether continuous NPC support is required to maintain hepatic functions), and whether this model system can provide a useful tool for drug development. In this dissertation, gene expression profiling, electroactive surface chemistry, and microfabrication tools are used to answer these questions. First, a functional genomic approach utilizing gene expression profiling was developed to identify and validate molecular mediators that modulate liver-specific functions in co-cultures. Second, the role of one candidate, T-cadherin, was characterized in detail. T- cadherin was found to upregulate hepatocyte functions in vitro upon presentation in both cellular (CHO cells) and acellular (immobilized protein) contexts. Third, micropatterned electroactive substrates were used to selectively release fibroblasts at various time points from co-culture to probe the dynamics of cell-cell interaction. Results indicated that continuous fibroblast stimulation was required to maintain hepatic functions. Finally, soft lithography tools were utilized to develop miniaturized, multi-well co-culture models of the human and rat liver that were stable for several weeks. Utility of microscale tissues for evaluating species-specific drug metabolism, drug-drug interactions, and susceptibility to a panel of hepatotoxins was demonstrated. These studies will have an impact on developing functional models of liver tissue for use in fundamental hepatology, cell-based therapies for liver disease and pharmaceutical drug development. Furthermore, the methods developed here can be generalized to other tissues where cell-cell interactions modulate cellular fate

Bioengineered Liver Models for Drug Testing and Cell Differentiation Studies

In vitro models of the human liver are important for the following: (1) mitigating the risk of drug-induced liver injury to human beings, (2) modeling human liver diseases, (3) elucidating the role of single and combinatorial microenvironmental cues on liver cell function, and (4) enabling cell-based therapies in the clinic. Methods to isolate and culture primary human hepatocytes (PHHs), the gold standard for building human liver models, were developed several decades ago; however, PHHs show a precipitous decline in phenotypic functions in 2-dimensional extracellular matrix–coated conventional culture formats, which does not allow chronic treatment with drugs and other stimuli. The development of several engineering tools, such as cellular microarrays, protein micropatterning, microfluidics, biomaterial scaffolds, and bioprinting, now allow precise control over the cellular microenvironment for enhancing the function of both PHHs and induced pluripotent stem cell–derived human hepatocyte-like cells; long-term (4+ weeks) stabilization of hepatocellular function typically requires co-cultivation with liver-derived or non–liver-derived nonparenchymal cell types. In addition, the recent development of liver organoid culture systems can provide a strategy for the enhanced expansion of therapeutically relevant cell types. Here, we discuss advances in engineering approaches for constructing in vitro human liver models that have utility in drug screening and for determining microenvironmental determinants of liver cell differentiation/function. Design features and validation data of representative models are presented to highlight major trends followed by the discussion of pending issues that need to be addressed. Overall, bioengineered liver models have significantly advanced our understanding of liver function and injury, which will prove useful for drug development and ultimately cell-based therapies