Enhancing the functional maturity of induced pluripotent stem cell–derived human hepatocytes by controlled presentation of cell–cell interactions in vitro

Abstract

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 ...

Deterministically patterned biomimetic human iPSC-derived hepatic model via rapid 3D bioprinting

“…In line with aggregate formation, the realignment of supporting cells along the hydrogel lines can potentially facilitate channel formation and possible vascularization (31). The greater HPC aggregate size observed in this 3D triculture model further supports the beneficial effects from supporting cells as widely reported in 2D coculture models (14,42).…”

“…hiPSC-derived hepatic cells, despite their potential to have malignant transformation following in vivo transplantations, have been widely recognized as the most promising candidate for developing patient-specific human hepatic models in vitro (4,7). Current in vitro liver models using hiPSC-derived hepatic cells are largely limited by their lack of biomimicry (3,7,(10)(11)(12)(13)(14). The 3D triculture model presented here highlights the successful application of DLP-based bioprinting technology to liver tissue engineering and thus the progress of the field to a level where the complex liver microarchitecture and cell composition can be studied in a physiologically relevant model.…”

“…Cells at both hepatic progenitor and maturation stages, i.e., HPCs and HLCs, respectively, have demonstrated their potentials in organizing into vascularized liver buds (11,13) and in the construction of in vitro models with simple geometry (10,12,14). To determine the stage of cells along the differentiation pathway that is more suitable for hydrogel encapsulation, HPCs and the relatively more mature HLCs were compared in terms of their albumin secretion time course following encapsulation in GelMA (Fig.…”

“…Major liver functions are tightly linked to the 3D assembly of hepatocytes with the supporting cell types from both endodermal and mesodermal origins in a hexagonal lobule unit. Current approaches to use HLCs for an in vitro liver model, however, are mostly limited to 2D culture or simple 3D spheroid cultures (3,7,(10)(11)(12)(13)(14). The lack of a biomimetic microenvironment provided from the 3D interactions of parenchymal and nonparenchymal cell types along the hepatic differentiation stages may potentially be one of the limiting factors to functional maturation of the hepatic progenitor cells (HPCs), as well as the functional preservation of HLCs in vitro (15,16).…”

Regenerative Medicine: Advances from Developmental to Degenerative Diseases

“…For example, phenotypes of hPSC-derived cardiomyocytes reflect structural, molecular and electrophysiology phenotypes of fetal, rather than fully mature adult cardiomyocytes 63,64 . Similarly, phenotypic and functional features of hPSC-derived hepatocytes 65 and  cells also appear to align more closely with their fetal rather than the adult counterparts 66 . Although fetal-like cells will undoubtedly prove useful in developmental studies and some aspects of disease modelling, cell replacement therapies necessitate the production of cells capable of generating fully functional adult cell types when coreceptor and costimulation (CD40L) blockade with monoclonal antibodies given at the time of transplantation 73 .…”