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The utility of in vitro human disease models is mainly dependent on the availability and functional maturity of tissue-specific cell types. We have previously screened for and identified small molecules that can enhance hepatocyte function in vitro. Here, we characterize the functional effects of one of the hits, FH1, on primary human hepatocytes in vitro, and also in vivo on primary hepatocytes in a zebrafish model. Furthermore, we conducted an analogue screen to establish the structure-activity relationship of FH1. We performed affinity-purification proteomics that identified NQO2 to be a potential binding target for this small molecule, revealing a possible link between inflammatory signaling and hepatocellular function in zebrafish and human hepatocyte model systems.

The utility of in vitro human disease models is mainly dependent on the availability and functional maturity of tissue-specific cell types. We have previously screened for and identified small molecules that can enhance hepatocyte function in vitro. Here, we characterize the functional effects of one of the hits, FH1, on primary human hepatocytes in vitro, and also in vivo on primary hepatocytes in a zebrafish model. Furthermore, we conducted an analogue screen to establish the structure-activity relationship of FH1. We performed affinity-purification proteomics that identified NQO2 to be a potential binding target for this small molecule, revealing a possible link between inflammatory signaling and hepatocellular function in zebrafish and human hepatocyte model systems.

Biologists increasingly use co-culture systems in which two or more cell types are grown in cell culture together in order to better model cells’ native microenvironments. Co-cultures are often required for cell survival or proliferation, or to maintain physiological functioning in vitro. Having two cell types co-exist in culture, however, poses several challenges, including difficulties distinguishing the two populations during analysis using automated image analysis algorithms. We previously analyzed co-cultured primary human hepatocytes and mouse fibroblasts in a high-throughput image-based chemical screen, using a combination of segmentation, measurement, and subsequent machine learning to score each cell as hepatocyte or fibroblast. While this approach was successful in counting hepatocytes for primary screening, segmentation of the fibroblast nuclei was less accurate. Here, we present an improved approach that more accurately identifies both cell types. Pixel-based machine learning (using the software ilastik) is used to seed segmentation of each cell type individually (using the software CellProfiler). This streamlined and accurate workflow can be carried out using freely available and open source software., National Science Foundation (U.S.) (NSF CAREER DBI 1148823), National Institutes of Health (U.S.) (NIH UH3 EB017103)

Malaria 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., Bill & Melinda Gates Foundation (Award 51066), Singapore. Agency for Science, Technology and Research (National Science Scholarship), National Institutes of Health (U.S.) (Grant UH3-EB017103), National Cancer Institute (U.S.) (Koch Institute Support (Core). Grant P30-CA14051)

Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2014., 203, Vita. Cataloged from PDF version of thesis., Includes bibliographical references (pages 139-150)., Chronic liver disease affects more than 500 million people worldwide. The only therapy shown to directly prevent mortality is organ transplantation. However, there is growing discrepancy between supply and demand of transplant-grade organs and transplant recipients are subject to a lifetime of immunosuppressive regimens. Therefore, the overall aim of this thesis is to advance alternatives to whole organ transplantation for liver diseases. Treating the failure of organs serving a multitude of biochemical functions, such as the liver, requires cell-based therapies. Such therapies should ideally employ human cells due to immunological concerns and the substantial differences between animal and human hepatocellular functions. Human cell lines, while renewable, lack the full functional capacity of primary adult hepatocytes, and for clinical applications there is a tumorigenic risk. Primary human hepatocytes have exhibited therapeutic potential; however, limited sourcing has been a bottleneck for many fields of research and clinical therapies. Pluripotent human stem cells are an attractive cell source, but to date, the hepatocyte-like cells obtain by directed differentiation continue to exhibit an immature hepatic phenotype, which resembles fetal hepatocytes more than adult hepatocytes. Motivated by these limitations, we report in the first section of this thesis, the development of a 384-well liver platform that incorporates cell-cell interactions to stabilize the hepatocyte phenotype, and enable high-throughput screening using cryopreserved primary human hepatocytes. We also developed attendant assays to assess cell fates in this platform through an automated image-based proliferation assay and an ELISA-based functional assay. In the second section of this thesis, we use the high-throughput liver platform to conduct a chemical screen of 12,480 small molecules. We identified 12 bioactive factors in three classes: small molecules that enhanced proliferation (PH), or funct, by Jing (Meghan) Shan., Ph. D.

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)

Cell-based therapies hold the potential to alleviate the growing burden of liver diseases. Such therapies require human hepatocytes, which, within the stromal context of the liver, are capable of many rounds of replication. However, this ability is lost ex vivo, and human hepatocyte sourcing has limited many fields of research for decades. Here we developed a high-throughput screening platform for primary human hepatocytes to identify small molecules in two different classes that can be used to generate renewable sources of functional human hepatocytes. The first class induced functional proliferation of primary human hepatocytes in vitro. The second class enhanced hepatocyte functions and promoted the differentiation of induced pluripotent stem cell–derived hepatocytes toward a more mature phenotype than what was previously obtainable. The identification of these small molecules can help address a major challenge affecting many facets of liver research and may lead to the development of new therapeutics for liver diseases., Broad Institute of MIT and Harvard (Scientific Planning and Allocation of Resources Committee Grant), National Institutes of Health (U.S.) (Grant NIH R01-DK065152), National Institutes of Health (U.S.) (Grant NIH R01-DK56966)