School of Biomedical Engineering, Colorado State University, Fort Collins, CO.
Hepatology. 2015 Apr;61(4):1370-81. doi: 10.1002/hep.27621.
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 drug-induced hepatotoxicity.
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.
诱导多能干细胞衍生的人肝样细胞（iHeps）可为研究人类肝脏发育和疾病的机制、在不同患者中测试药物的疗效和安全性（即个性化医疗）以及在临床上实现基于细胞的治疗提供有力工具。然而，目前依赖生长因子和细胞外基质（ECM）的体外方案仅产生相对于成人原代人肝细胞（PHHs）肝脏功能水平较低的 iHeps。此外，这些 iHeps 中的低肝脏功能很难在培养中长期维持（数周到数月）。在这里，我们设计了一种微图案共培养（iMPCC）平台，采用多孔格式，与传统的致密培养相比，当与多个 PHH 供体进行基准比较时，iMPCC 显著增强了 iHep 在体外培养中的功能成熟和长期培养能力，至少可达 4 周。具体而言，iHeps 被微图案化到经验优化尺寸的胶原涂层区域上，周围是 3T3-J2 鼠胚胎成纤维细胞，然后用一层薄的 ECM 凝胶（Matrigel）夹在中间。我们通过全局基因表达谱、肝极性、白蛋白和尿素的分泌、基础细胞色素 P450（CYP450）活性、二期结合、药物介导的 CYP450 诱导以及药物诱导的肝毒性来评估 iHep 的成熟度。
控制 iHep 之间的同质相互作用和与基质成纤维细胞之间的异质相互作用显著成熟了 iHep 的功能，并使其在培养中维持数周。在未来，iMPCC 可能有助于药物筛选、研究 iHep 分化的分子机制、模拟肝脏疾病以及整合到旨在评估化合物对多器官反应的人芯片系统中。
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