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  • A common shortcoming of iHLCs and other iPSC derived


    A common shortcoming of iHLCs and other iPSC-derived cloprostenol lies in the fact that they often resemble a developmentally immature state compared to the fully differentiated adult counterpart (Engle and Puppala, 2013). In the field of iHLCs, current in vitro differentiation protocols result in the production of a hepatic cell type that is biologically closer to fetal hepatocytes than adult hepatocytes, due to the incomplete abrogation of the expression of fetal markers such as alpha-fetal protein (AFP) (Si-Tayeb et al., 2010; Song et al., 2009), and the incomplete acquisition of an adult-like levels of key secretory, detoxification, and metabolic activity (Shan et al., 2013). Our data show that further chemical maturation of iHLCs allows the acquisition of primaquine sensitivity, presumably via the acquisition of a drug metabolism enzyme (DME) expression profile that better resembles the adult human hepatocyte. This advance decreases the biological gap between iHLCs and primary adult hepatocytes and increases the potential utility of iHLCs in drug development efforts for malaria and other diseases. The expression of a developmentally mature repertoire of hepatic DMEs is particularly important considering that current drug development efforts toward malaria eradication revolves around the 8-aminoquinoline family, which is currently the only class of drugs that is efficacious against the cryptic hypnozoite stage of P. vivax liver-stage infections (Wells et al., 2009), cloprostenol and the fact that many existing antimalarial drugs such as proguanil, artemether, lumefantrine, and halofantrine are known to undergo metabolism in the liver by hepatic DMEs (Khoo et al., 2005) and whose in vitro efficacy will therefore likely be predictive of in vivo efficacy only if the in vitro hepatocyte model exhibits a primary human hepatocyte DME expression profile. Although the primaquine response in iHLCs pretreated with FPH1 was incomplete, other primary human hepatocyte models of P. falciparum treated with the same primaquine concentration (10 μM) also exhibit an incomplete response, with about 10%–20% of the number of P.f. EEFs remaining (Dembele et al., 2011; March et al., 2013). An unexpected finding was that FPH1 pretreatment also increased the baseline number of P.f. EEFs in iHLCs in the absence of primaquine treatment compared to DMSO pretreatment (Figure 5C). The chemical maturation of iHLCs by FPH1 is likely to involve a complex mechanism, which could theoretically result either in an increase in the adult hepatic phenotype of the existing population of iHLCs and hence their infectibility by Plasmodium sporozoites, or an expansion in the population of iHLCs or of other hepatic progenitors. Chemically matured iHLCs with increased baseline infectibility with Plasmodium could therefore provide an opportunity to elucidate hepatic host factors that promote liver-stage malaria infection in hepatocytes. In conclusion, the establishment of Plasmodium liver-stage infections in induced pluripotent stem cell-derived hepatocyte-like cells lays the foundation for their use in antimalarial drug discovery as well as paves the way to study the genetic basis of host-Plasmodium interactions.
    Experimental Procedures
    Author Contributions
    Acknowledgments We thank Ana Rodriguez and Sandra Gonzalez (New York University) for providing mosquitoes infected with P. yoelii and P. berghei, Dyann Wirth and Emily Lund (Harvard School of Public Health) for providing mosquitoes infected with P. berghei, Photini Sinnis and Abhai Tripathi (Johns Hopkins Bloomberg School of Public Health) for providing mosquitoes infected with P. falciparum, Jetsumon Sattabongkot Prachumsri and Rapatbhorn Patrapuvich (Mahidol University) for providing fresh P. vivax sporozoites, Cellular Dynamics International (CDI) for providing iCell Hepatocytes 2.0 for P. vivax experiments, Jeffrey B. Wyckoff (Koch Institute, MIT) for technical help in confocal microscopy, Stephen Duncan (Medical College of Wisconsin) for iPS cell lines and counsel on iHep generation, and Heather Fleming for critical reading and help with manuscript preparation. This work was supported by the Bill and Melinda Gates Foundation (award number 51066). S.N. is supported by an , US National Institutes of Health (NIH) grant UH3-EB017103, and in part by the Koch Institute Support (core) Grant P30-CA14051 from the National Cancer Institute. S.N.B. is a Howard Hughes Medical Institute Investigator.