Induced pluripotent stem cell-derived human hepatocyte-like cells (iHeps) could provide a powerful tool for studying the mechanisms underlying human liver development and disease, testing the efficacy and safety of pharmaceuticals across different patients (i.e., personalized medicine), and enabling cell-based therapies in the clinic. However, current in vitro protocols that rely upon growth factors and extracellular matrices (ECMs) alone yield iHeps with low levels of liver functions relative to adult primary human hepatocytes (PHHs). Moreover, these low hepatic functions in iHeps are difficult to maintain for prolonged times (weeks to months) in culture. Here, we engineered a micropatterned coculture (iMPCC) platform in a multiwell format that, in contrast to conventional confluent cultures, significantly enhanced the functional maturation and longevity of iHeps in culture for at least 4 weeks in vitro when benchmarked against multiple donors of PHHs. In particular, iHeps were micropatterned onto collagen-coated domains of empirically optimized dimensions, surrounded by 3T3-J2 murine embryonic fibroblasts, and then sandwiched with a thin layer of ECM gel (Matrigel). We assessed iHep maturity by global gene expression profiles, hepatic polarity, secretion of albumin and urea, basal cytochrome P450 (CYP450) activities, phase II conjugation, drug-mediated CYP450 induction, and druginduced hepatotoxicity. Conclusion: Controlling both homotypic interactions between iHeps and heterotypic interactions with stromal fibroblasts significantly matures iHep functions and maintains them for several weeks in culture. In the future, iMPCCs could prove useful for drug screening, studying molecular mechanisms underlying iHep differentiation, modeling liver diseases, and integration into human-on-a-chip systems being designed to assess multiorgan responses to compounds. (HEPATOLOGY 2015;61:1370-1381 O wing to significant species-specific differences in liver pathways, in vitro models of the human liver play an important role in drug development and mechanistic investigations. 1,2 Isolated primary human hepatocytes (PHHs) are ideal for constructing such models because they can maintain high levels of key liver functions for several weeks in vitro under specific culture conditions. 1,3-6 However, PHHs are a severely limited resource given shortages in donor livers, and their quality for in vitro use can vary considerably across different cell lots. Induced pluripotent stem cell-derived human hepatocyte-like cells (iHeps)Abbreviations: AFP, alpha-fetoprotein; ALB, albumin; ARG1, arginase 1; CDF, 5-(and-6)-carboxy-2 0 ,7 0 -dichlorofluorescein diacetate; CDI, Cellular Dynamics International (Madison, WI); CYP450, cytochrome P450; DAPI, 4 0 ,6-diamidino-2-phenylindole; DDI, drug-drug interactions; ECMs, extracellular matrices; FDA, U.S. Food and Drug Administration; GRNs, gene regulatory networks; HNF6, hepatocyte nuclear factor 6; iCCs, conventional confluent cultures of iHeps with Matrigel overlay; iHeps, induced pluripotent stem ...

Primary human hepatocytes (PHHs) are a limited resource for drug screening, their quality for in vitro use can vary considerably across different lots, and a lack of available donor diversity restricts our understanding of how human genetics affect drug-induced liver injury (DILI). Induced pluripotent stem cell-derived human hepatocyte-like cells (iPSC-HHs) could provide a complementary tool to PHHs for high-throughput drug screening, and ultimately enable personalized medicine. Here, we hypothesized that previously developed iPSC-HH-based micropatterned co-cultures (iMPCCs) with murine embryonic fibroblasts could be amenable to long-term drug toxicity assessment. iMPCCs, created in industry-standard 96-well plates, were treated for 6 days with a set of 47 drugs, and multiple functional endpoints (albumin, urea, ATP) were evaluated in dosed cultures against vehicle-only controls to enable binary toxicity decisions. We found that iMPCCs correctly classified 24 of 37 hepatotoxic drugs (65% sensitivity), while all 10 non-toxic drugs tested were classified as such in iMPCCs (100% specificity). On the other hand, conventional confluent cultures of iPSC-HHs failed to detect several liver toxins that were picked up in iMPCCs. Results for DILI detection in iMPCCs were remarkably similar to published data in PHH-MPCCs (65% versus 70% sensitivity) that were dosed with the same drugs. Furthermore, iMPCCs detected the relative hepatotoxicity of structural drug analogs and recapitulated known mechanisms of acetaminophen toxicity in vitro. In conclusion, iMPCCs could provide a robust tool to screen for DILI potential of large compound libraries in early stages of drug development using an abundant supply of commercially available iPSC-HHs.

Drug-induced liver injury (DILI) is a leading cause of drug attrition. Significant and well-documented differences between animals and humans in liver pathways now necessitate the use of human-relevant in vitro liver models for testing new chemical entities during preclinical drug development. Consequently, several human liver models with various levels of in vivo-like complexity have been developed for assessment of drug metabolism, toxicity, and efficacy on liver diseases. Recent trends leverage engineering tools, such as those adapted from the semiconductor industry, to enable precise control over the microenvironment of liver cells and to allow for miniaturization into formats amenable for higher throughput drug screening. Integration of liver models into organs-on-a-chip devices, permitting crosstalk between tissue types, is actively being pursued to obtain a systems-level understanding of drug effects. Here, we review the major trends, challenges, and opportunities associated with development and implementation of engineered liver models created from primary cells, cell lines, and stem cell-derived hepatocyte-like cells. We also present key applications where such models are currently making an impact and highlight areas for improvement. In the future, engineered liver models will prove useful for selecting drugs that are efficacious, safer, and, in some cases, personalized for specific patient populations.

Drug-induced liver injury (DILI) remains a leading cause of drug withdrawal from human clinical trials or the marketplace. Due to species-specific differences in liver pathways, predicting human-relevant DILI using in vitro human liver models is critical. Microfabrication tools allow precise control over the cellular microenvironment towards stabilizing liver functions for weeks. These tools are used to engineer human liver models with different complexities and throughput using cell lines, primary cells, and stem cell-derived hepatocytes. Including multiple human liver cell types can mimic cell-cell interactions in specific types of DILI. Finally, organ-on-a-chip models demonstrate how drug metabolism in the liver affects multi-organ toxicities. In this review, we survey engineered human liver platforms within the needs of different phases of drug development.