A Microscale Human Liver Platform that Supports the Hepatic Stages of Plasmodium falciparum and vivax

The Plasmodium liver stage is an attractive target for the development of antimalarial drugs and vaccines, as it provides an opportunity to interrupt the life cycle of the parasite at a critical early stage. However, targeting the liver stage has been difficult. Undoubtedly, a major barrier has been the lack of robust, reliable, and reproducible in vitro liver-stage cultures. Here, we establish the liver stages for both Plasmodium falciparum and Plasmodium vivax in a microscale human liver platform composed of cryopreserved, micropatterned human primary hepatocytes surrounded by supportive stromal cells. Using this system, we have successfully recapitulated the full liver stage of P. falciparum, including the release of infected merozoites and infection of overlaid erythrocytes, as well as the establishment of small forms in late liver stages of P. vivax. Finally, we validate the potential of this platform as a tool for medium-throughput antimalarial drug screening and vaccine development.Bill & Melinda Gates Foundation (51066

Engineering human hepatic tissue for modeling liver-stage malaria

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2014.Cataloged from PDF version of thesis. Vita.Includes bibliographical references (pages 132-153).The Plcsmodium liver stage is an attractive target for the development of antimalarial drugs and vaccines, as it provides an opportunity to interrupt the life cycle of the parasite at a critical early stage. However, targeting the liver stage has been difficult due to a lack of human liver models that robustly recapitulate host-pathogen interactions in a physiologically relevant cell type. Through the application of hepatic tissue engineering concepts and techniques, this thesis sought to develop advanced models of liver-stage malaria that will allow the facile interrogation of potential antimalarial drugs in primary human hepatocytes. In the first part of this work, we established liver-stage Plasmodium infection in an engineered microscale human liver platform based on micropatterned cocultures of primary human hepatocytes and supportive stromal cells, enabling medium-throughput phenotypic screens for potential antimalarial drugs in a more authentic host cell, and demonstrated the utility of this model for malaria vaccine testing. We further hypothesized and showed that recapitulation of a more physiologically relevant oxygen tension that is experienced by hepatocytes in vivo improved infection rates and parasite growth in vitro. Next, we demonstrated the feasibility of establishing liver-stage malaria infections in human induced pluripotent stem cell-derived hepatocyte-like cells (iHLCs), thus enabling the study of host genetic variation on liver-stage malaria infection and antimalarial drug responses. We also applied recently discovered small molecules to induce further hepatic maturation, thus increasing the utility of using iHLCs for antimalarial drug development. Finally, we designed and provided a proof-of-concept for a humanized mouse model of liver-stage malaria that involves the fabrication and ectopic implantation of PEG-cryogel-based engineered human artificial livers, and can be generated in a facile, rapid and scalable fashion for future preclinical antimalarial drug testing in vivo. The results of this research represent a three-pronged approach towards engineering scalable human liver models that recapitulate liver-stage malaria infection which may ultimately facilitate antimalarial drug discovery at various stages of the drug development pipeline.by Shengyong Ng.Ph. D

In vitro alterations do not reflect a requirement for host cell cycle progression during Plasmodium liver stage infection

Copyright © 2015, American Society for Microbiology. All Rights Reserved.Prior to invading nonreplicative erythrocytes, Plasmodium parasites undergo their first obligate step in the mammalian host inside hepatocytes, where each sporozoite replicates to generate thousands of merozoites. While normally quiescent, hepatocytes retain proliferative capacity and can readily reenter the cell cycle in response to diverse stimuli. Many intracellular pathogens, including protozoan parasites, manipulate the cell cycle progression of their host cells for their own benefit, but it is not known whether the hepatocyte cell cycle plays a role during Plasmodium liver stage infection. Here, we show that Plasmodium parasites can be observed in mitotic hepatoma cells throughout liver stage development, where they initially reduce the likelihood of mitosis and ultimately lead to significant acquisition of a binucleate phenotype. However, hepatoma cells pharmacologically arrested in S phase still support robust and complete Plasmodium liver stage development, which thus does not require cell cycle progression in the infected cell in vitro. Furthermore, murine hepatocytes remain quiescent throughout in vivo infection with either Plasmodium berghei or Plasmodium yoelii, as do Plasmodium falciparum-infected primary human hepatocytes, demonstrating that the rapid and prodigious growth of liver stage parasites is accomplished independent of host hepatocyte cell cycle progression during natural infection.This work was supported by funding from the European Research Council under the European Union's 7th Framework Programme (FP7/2007-2013) through ERC grant agreement number 311502 (M.M.M.) and from Fundação para a Ciência e Tecnologia (FCT; Portugal) through grant number EXCL/IMI-MIC/0056/2012 (M.M.M.). K.K.H. was supported by funds from the European Community's Seventh Framework Programme (FP7/2007-2013) Marie Cure IEF (PIEF-GA-2008-221854) and Fundação para a Ciência e Tecnologia (SFRH/BPD/40989/2007).info:eu-repo/semantics/publishedVersio

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

SummaryMalaria 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

Towards a Humanized Mouse Model of Liver Stage Malaria Using Ectopic Artificial Livers

The malaria liver stage is an attractive target for antimalarial development, and preclinical malaria models are essential for testing such candidates. Given ethical concerns and costs associated with non‐human primate models, humanized mouse models containing chimeric human livers offer a valuable alternative as small animal models of liver stage human malaria. The best available human liver chimeric mice rely on cellular transplantation into mice with genetically engineered liver injury, but these systems involve a long and variable humanization process, are expensive, and require the use of breeding-challenged mouse strains which are not widely accessible. We previously incorporated primary human hepatocytes into engineered polyethylene glycol (PEG)-based nanoporous human ectopic artificial livers (HEALs), implanted them in mice without liver injury, and rapidly generated human liver chimeric mice in a reproducible and scalable fashion. By re-designing the PEG scaffold to be macroporous, we demonstrate the facile fabrication of implantable porous HEALs that support liver stage human malaria (P. falciparum) infection in vitro, and also after implantation in mice with normal liver function, 60% of the time. This proof-of-concept study demonstrates the feasibility of applying a tissue engineering strategy towards the development of scalable preclinical models of liver stage malaria infection for future applications.Bill & Melinda Gates Foundation (Global Health Grant initiative (Global Health Grant: OPP1023607))National Cancer Institute (U.S.) (Grant P30-CA14051)National Institute of Environmental Health Sciences (Core Center Grant P30-ES002109)Singapore. Agency for Science, Technology and Research (National Science Scholarship)