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In this study we show the feasibility of infecting
In this study, we show the feasibility of infecting iHLCs with Plasmodium sporozoites in vitro and demonstrate Plasmodium parasite development over time in culture. We identify the stage at which etizolam vendor acquire permissiveness to liver-stage malaria infection during the differentiation process. It is also necessary to characterize the responses of malaria-infected iHLCs to known antimalarial drugs in order to establish the utility of this cell type for use in in vitro liver-stage malaria phenotypic drug screens. We observe that iHLCs are not responsive to the antimalarial drug primaquine and hypothesize this deficiency is due to a lack of bioactivation of the drug by hepatic cytochrome P450 drug metabolism enzymes. Consistent with this model, we further demonstrate that chemically matured iHLCs acquire primaquine sensitivity, highlighting the potential to use iHLCs for antimalarial drug testing.
Results
Discussion
In this study, we show the feasibility of infecting iHLCs with P. berghei, P. yoelii, P. falciparum, and P. vivax. Liver-stage Plasmodium EEFs grow in size over time and express MSP-1, which is typically expressed in more mature EEFs. Although iPS cells and definitive endoderm cells are generally not infectible with P. falciparum, some degree of P. falciparum infectibility is acquired by the time the cells are further differentiated to a hepatic-specified endoderm lineage. Notably, populations of hepatoblasts are equally or more infectible than the resulting iHLCs at the end of the in vitro differentiation protocol. iHLCs generated by the existing differentiation protocol do not respond to primaquine, a malaria drug that requires bioactivation by mature hepatic cytochrome P450 (CYP450) enzymes. However, further maturation of iHLCs using a previously described small molecule results in the acquisition of primaquine sensitivity, such that the drug treatment diminished infection by P. yoelii and P. falciparum.
In the context of drug development, there has been a shift from the traditional paradigm of testing drugs in immortalized cell lines to the use of primary cells, which have been increasingly recognized to offer better physiological relevance to drug screening and disease modeling in vitro (Engle and Puppala, 2013). A key goal of early-stage drug discovery platforms is the elimination of drug candidates that generate toxic metabolites that can cause drug-induced liver injury (DILI), a key cause of drug removal from the market (McDonnell and Braverman, 2006). To this end, a hepatic cell type that accurately recapitulates the native cellular physiology of an adult human hepatocyte is advantageous, but most hepatic cell lines lack the expression of a wide array of such key adult hepatic metabolism activity (March et al., 2013) because they are largely tumor derived (i.e., HepG2) or tumor associated (i.e., HC04). A second major goal of drug discovery platforms is the ability to identify nontoxic compounds that demonstrate differential efficacy in a phenotypic screen relevant to a disease. In the case of drug discovery against the malarial liver stages, it is therefore highly advantageous to utilize a cell type that best represents the primary adult hepatocyte. At the same time, it is also ideal to represent highly polymorphic genetic variants in drug metabolism and different ethnic groups in such drug screens, as these factors may influence the efficacy of potential antimalarial drug candidates. For example, genetic polymorphisms in CYP2D6 metabolism that stratified P. vivax patients into poor, intermediate, or extensive CYP2D6 metabolizers were recently reported to correlate with the risk of a failure of primaquine to prevent malaria relapse due to P. vivax (Bennett et al., 2013). Several cell sources have been proposed to augment the genetic variation and supply of adult primary human hepatocytes, including xenogenic or fetal human tissue, or embryonic stem cell-derived hepatocytes, but these sources are hampered by safety, ethical, or sourcing issues.