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 ...
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