Jing Shan

Human iPSC-Derived Hepatocyte-like Cells Support Plasmodium Liver-Stage Infection In Vitro

Summary Malaria eradication is a major goal in public health but is challenged by relapsing malaria species, expanding drug resistance, and the influence of host genetics on antimalarial drug efficacy. To overcome these hurdles, it is imperative to establish in vitro assays of liver-stage malaria for drug testing. Induced pluripotent stem cells (iPSC) potentially allow the assessment of donor-specific drug responses, and iPSC-derived hepatocyte-like cells (iHLCs) can facilitate the study of host genetics on host-pathogen interactions and the discovery of novel targets for antimalarial drug development. We establish in vitro liver-stage malaria infections in iHLCs using P. berghei, P. yoelii, P. falciparum, and P. vivax and show that differentiating cells acquire permissiveness to malaria infection at the hepatoblast stage. We also characterize antimalarial drug metabolism capabilities of iHLCs using prototypical antimalarial drugs and demonstrate that chemical maturation of iHLCs can improve their potential for antimalarial drug testing applications.

High-Throughput Platform for Identifying Molecular Factors Involved in Phenotypic Stabilization of Primary Human Hepatocytes In Vitro

Liver disease is a leading cause of morbidity worldwide and treatment options are limited, with organ transplantation being the only form of definitive management. Cell-based therapies have long held promise as alternatives to whole-organ transplantation but have been hindered by the rapid loss of liver-specific functions over a period of days in cultured hepatocytes. Hypothesis-driven studies have identified a handful of factors that modulate hepatocyte functions in vitro, but our understanding of the mechanisms involved remains incomplete. We thus report here the development of a high-throughput platform to enable systematic interrogation of liver biology in vitro. The platform is currently configured to enable genetic knockdown screens and includes an enzyme-linked immunosorbent assay–based functional assay to quantify albumin output as a surrogate marker for hepatocyte synthetic functions as well as an image-based viability assay that counts hepatocyte nuclei. Using this platform, we identified 12 gene products that may be important for hepatocyte viability and/or liver identity in vitro. These results represent important first steps in the elucidation of mechanisms instrumental to the phenotypic maintenance of hepatocytes in vitro, and we hope that the tools reported here will empower additional studies in various fields of liver research.

Hepatic Tissue Engineering

Liver tissue engineering aims to provide novel therapies for liver diseases and create effective tools for understanding fundamental aspects of liver biology and pathologic processes. Approaches range from bio-mimetic in vitro model systems of the liver to three-dimensional implantable constructs. Collectively, these cell-based approaches endeavor to replace or enhance organ transplantation, which is the current standard treatment for liver diseases in most clinical settings. However, the complexity of liver structure and function as well as the limited supply of human hepatocytes pose unique challenges for the field. This chapter reviews advances in the field of liver tissue engineering within the context of current therapies for liver diseases, and clinical alternatives such as cell transplantation strategies and extracorporeal bioartificial liver devices.

Chapter 46 – Hepatic Tissue Engineering

Cell-based therapies for liver failure offer the potential to augment or replace whole organ transplantation. However, the development of such therapies poses unique challenges stemming from both the complexity of liver structure and function as well as the fact that the liver is the largest internal organ in the body. The field of liver tissue engineering encompasses several approaches aimed collectively at providing novel therapeutic options for liver disease patients as well as elucidating fundamental aspects of hepatic biology. These approaches include the development of: (1) in vitro model systems that recapitulate normal liver function, (2) extracorporeal bioartifical liver devices for the temporary support of liver failure patients, and (3) three-dimensional (3D) implantable constructs for both human therapy and ‘humanized’ animal models. Advances in each of these areas are reviewed in this chapter within the context of current treatments for liver disease and additional clinical alternatives such as surgical advancements for organ transplant and cell transplantation strategies.

762 Control of Microenvironmental Cues Enables the Differentiation of Pluripotent Stem Cell Derived Hepatocyte-Like Cells to an Adult Phenotype

Biliary atresia (BA) is a progressive fibro-inflammatory cholangiopathy affecting the intraand extrahepatic bile ducts of neonates. Although BA is the most common identifiable cause of obstructive jaundice in infants, and is the leading indication for pediatric liver transplantation, the etiology remains elusive. We investigated the importance of genes identified in genome-wide association studies (GWAS) of BA patients using zebrafish. The Matthewss lab has demonstrated the utility of the zebrafish system to test the functionality of genes identified in GWAS of BA by demonstrating that knockdown of gpc1 leads to biliary defects. A prior GWAS examined single-nucleotide polymorphisms in 200 Han Chinese BA patients and 481 ethnically matched controls. The strongest association was found for a region located between the XPNPEP1 and ADD3 genes on 10q24.2. Our genetic analysis confirmed the importance of this region in a separate cohort of patients. To determine whether loss of xpnpep1 and/or add3a leads to biliary defects we performed knockdown studies of the respective genes using morpholino antisense oligonucleotides (MO). We then examined their biliary function using the lipid reporter PED6, biliary development using cytokeratin immunostaining, and gene expression using quantitative PCR. We confirmed that xpnpep1 and add3a are expressed in the developing zebrafish liver. Knockdown of add3a led to decreased biliary function by PED6 screening, while xpnpep1 knockdown had only a mild effect. There were developmental biliary defects in the add3a morphants, as demonstrated by cytokeratin immunostaining. The transcription factor vhnf1, implicated in biliary development, was significantly downregulated in add3a morphants but not xpnpep1 morphants. add3a morphants also demonstrated increased expression of gli2a, a Hedgehog target, which is consistent with prior studies of BA patients and of zebrafish models of BA such as gpc1 knockdown. Interestingly, knockdown of both add3a and gpc1 resulted in a synergistic disruptive effect on biliary development. While GWAS identified ADD3 and XPNPEP1 as potential BA susceptibility genes, our results suggest that ADD3 is likely the more important gene in this regard. Like gpc1, add3a acts via Hedgehog signaling, supporting a role for this important pathway in BA pathogenesis.