Modeling Human Disease by Interacting with Pathogens
Many virus and parasite pathogens specifically target liver cells, and these 'hepatotropic' infections affect over 500 million people worldwide. These pathogens include Hepatitis B and C and the liver stages of Malaria (caused by Plasmodium falciparum and vivax). Of the over 200 cell types in the body, these pathogens exclusively infect hepatocytes – and only from human or other closely related species, which means that any preclinical studies are limited to primates or humanized mice. There are human cell lines that have been shown to be infectible by these viruses and parasites, and have been used in the past to study the pathogens and to test candidate drugs and vaccines. However, these cell lines don't behave like normal human liver cells in many important ways, and therefore it is difficult to be confident that results obtained using them will be consistent with what occurs in a patient. Primary human hepatocytes, isolated directly from human liver samples, are considered a more relevant system because the cells are closer to normal human biology, but it is very challenging to maintain these cells outside of the liver. In our group, we used micropatterned co-cultures (MPCCs) to keep primary human hepatocytes alive in culture, and they behave much like liver cells do before being removed from the body (Khetani & Bhatia, Nat Biot 2008). Liver cells grown in MPCCs express proteins on their surface that serve as the 'entry receptor' for HCV, and infected MPCCs can support the full viral life cycle for several weeks (Ploss et al., PNAS 2010). When combined with a fluorescent- and luminescent-based tool that allows us to track virus activity based on light emission (in collaboration with Charles Rice, Rockefeller), we demonstrated that MPCCs can be used to compare antibodies for their ability to block viral infection, and to test the efficacy and toxicity of antiviral drug candidates. small molecules, and their interdependence (Andrus et al., Hepatology 2011). MPCCs also support the full liver-stage life cycle of two forms of human Malaria, including the appearance of persistent 'dormant' small forms of Plasmodium vivax that represent a key barrier to the newly-announced malaria eradication campaign (March et al. CHM 2013). Using frozen cells from human donors, we have uncovered that liver cells from different individuals vary in their capacity to be infected by these pathogens. We also established the potential for 'personalized' models of hepatotropic infection by adapting iHeps to HCV (Schwartz/Trehan, PNAS 2012), enabling the modeling of rare clinical phenotypes through reprogramming. Collectively, these tools provide the first examples of using engineered human tissues for infectious disease modeling and are rapidly becoming adopted in the field.
MPCCs as a modeling platform for hepatotropic pathogens. (A) Micrograph of MPCC platform (scale bar: 200 um; H: primary human hepatocytes, F: fibroblasts). (B) MPCCs recapitulate HCV life cycle (top). HCV luciferase reporter (lower left) confirms persistent HCV replication not seen in conventional liver models. MPCCs express HCV entry receptors (IF staining for CD81, scale bar: 20mM); neutralizing antibodies show that blocking various epitopes inhibits HCV entry with different potencies, as listed. (C) P.falciparum (left) and vivax (right) infection in MPCC, showing persistent P.v. at 21 days. (D) Heat map displays of human-specific drug metabolism genes and IC50 of primaquine in MPCC vs hepatoma, HC04. (E) Effect of cell surface pO2 on Plasmodium infection. Scale bar: 5µm (C,E) Khetani et al., 2008; Ploss et al., 2010; March et al., 2013; Ng et al., 2014)