iPSC derived iHLCs offer a unique

iPSC-derived iHLCs offer a unique advantage in that they can be generated from any donor, which allows a broad spectrum of the human population to be represented in in vitro drug screens and provides an opportunity to assess donor-specific drug responses in vitro. Recent collaborations between drug discovery companies and academic laboratories (Engle and Puppala, 2013) mark the development of an infrastructure that will benefit future personalized models of human diseases, including hepatotropic diseases like the relapsing forms of malaria. iPSCs can also be engineered using DNA editing techniques to incorporate any genetic abnormality or modification to enable the exploration of the role that host gp120 plays in liver-stage malaria infection. Although clinically relevant host factors that influence hepatic susceptibility to liver-stage malaria infection have not been documented, the study of potential host factors may benefit from the larger pool of genetic variation that is accessible with the use of iPSCs. This effort may have implications in drug discovery against the liver stages of malaria species that present with large genetic heterogeneities in different parts of the world, especially the relapsing malaria species like P. vivax (e.g., temperate versus tropical strains), which may have coevolved to infect geographically distinct human populations (Cui et al., 2003; Dondorp et al., 2009; Gunawardena et al., 2010; Li et al., 2001). From a developmental standpoint, the infectibility of iHLCs with liver-stage malaria and other hepatotropic pathogens such as the hepatitis viruses could also be a potential biomarker of hepatic lineage commitment, with iPSCs becoming permissive only when they become sufficiently hepatocyte-like. The ability to study liver-stage malaria infection at different developmental stages during the in vitro directed differentiation of iPSCs into iHLCs within a single donor may also facilitate the identification of host factors required for permissiveness to Plasmodium sporozoite infection and the elucidation of the roles that these host factors play in malaria pathogenesis. One potential measure of how developmentally close iHLCs are to primary human hepatocytes is the efficiency of liver-stage malaria infection of iHLCs compared to primary human hepatocytes. With the multiplicity of infection (MOI) of approximately 0.5 used in the above P. falciparum experiments (with respect to the estimated number of iHLCs per well based on an estimate of differentiation efficiency), an infection rate of approximately 0.3–1.8 P.f. EEFs per 10,000 iHLCs is obtained. In comparison, a microscale human liver platform that was recently reported to support P. falciparum infection in primary human hepatocytes reported an infection rate of approximately 100 P.f. EEFs per 10,000 hepatocytes at 3.5 days after infection with an MOI of 15 (March et al., 2013). Assuming that the infection rate scales linearly with MOI for the purposes of this analysis, an MOI of 0.5 in primary human hepatocytes would be estimated to give rise to an infection rate of approximately 3 P.f. EEFs per 10,000 hepatocytes. This suggests that iHLCs exhibit anywhere from 10%–60% of the infectibility of primary human hepatocytes and indicates that iHLCs have acquired sufficient hepatocyte-like characteristics that confer infectibility with liver-stage malaria. The observation that iHLCs are infectible with malaria before their in vitro differentiation is complete could reflect the acquisition of some liver-stage malaria host entry factors during the iPSC differentiation process, or heterogeneity in iPSC differentiation that results in faster hepatic maturation of a subpopulation of cells. This observation could also suggest the possibility of some promiscuity in the host entry factors that are required for Plasmodium sporozoite entry. Further studies are required to determine whether the EEFs observed in incompletely differentiated iPSCs reflect fully replication-competent EEFs, or whether such EEFs are prone to developmental arrest due to the absence (or presence) of some host factor that promotes (or inhibits) parasite development. It is intriguing that hepatoblasts appear to support similar P. falciparum infection rates and similar degrees of parasite growth as iHLCs, with some experiments even exhibiting a trend of increased numbers of P.f. EEFs or P.y. EEFs in hepatoblasts compared to iHLCs (Figures 3B, left, and S2E). This slight difference in infectibility could suggest the acquisition of sufficient host entry factors that support Plasmodium infection and development by d15 postinitiation of differentiation from iPSCs, but that further maturation beyond the hepatoblast stage results in the acquisition of other host factors that henceforth limit EEF development and survival. Because the cells in different stages of in vitro differentiation arise from the same donor, these observations also offer a clean comparative system in which to systematically probe and identify host factors that are essential for liver-stage malaria parasite infections, using proteomics or gene expression technologies, in a donor-independent manner.