An Efficient Method for Differentiation of Human Induced Pluripotent Stem Cells into Hepatocyte-like Cells Retaining Drug Metabolizing Activity

Kondo, Yuki; Iwao, Takahiro; Nakamura, Katsunori; Sasaki, Takuma; Takahashi, Shogo; Kamada, Noboru; Matsubara, Tsutomu; Gonzalez, Frank J.; Akutsu, Hidenori; Miyagawa, Yoshitaka; Okita, Hajime; Kiyokawa, Nobutaka; Toyoda, Masashi; Umezawa, Akihiro; Nagata, Kiyoshi; Matsunaga, Tamihide; Ohmori, Shigeru

doi:10.2133/dmpk.dmpk-13-rg-104

Cited by 54 publications

(38 citation statements)

References 41 publications

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“…The hepatic differentiation protocol used in our study is similar to that in many reports (7,9). Our results of characterization of each differentiation stage by both immunofluorescent staining of key markers and expression levels of important genes are within the ranges of those observed in literature (7,9), suggesting that the differentiation process has been successful. The characterizations of the cells along differentiation stages and before encapsulation by staining ( Fig.…”

Section: Discussionsupporting

confidence: 84%

“…Many groups have reported monolayer differentiation of hiPSCs into hepatocyte-like cells (HLCs) and their ability to metabolize drugs (7)(8)(9). Nevertheless, hiPSC-derived HLCs are still considered immature in terms of many liver-specific gene expressions, functions, and cytochrome P450 (CYP) enzyme activities (7,9).…”

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confidence: 99%

“…Nevertheless, hiPSC-derived HLCs are still considered immature in terms of many liver-specific gene expressions, functions, and cytochrome P450 (CYP) enzyme activities (7,9). 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.…”

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confidence: 99%

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

Zhu

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

746

661

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The functional maturation and preservation of hepatic cells derived from human induced pluripotent stem cells (hiPSCs) are essential to personalized in vitro drug screening and disease study. 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. Although there are many reports on functional 2D cell differentiation, few studies have demonstrated the in vitro maturation of hiPSC-derived hepatic progenitor cells (hiPSC-HPCs) in a 3D environment that depicts the physiologically relevant cell combination and microarchitecture. The application of rapid, digital 3D bioprinting to tissue engineering has allowed 3D patterning of multiple cell types in a predefined biomimetic manner. Here we present a 3D hydrogel-based triculture model that embeds hiPSC-HPCs with human umbilical vein endothelial cells and adiposederived stem cells in a microscale hexagonal architecture. In comparison with 2D monolayer culture and a 3D HPC-only model, our 3D triculture model shows both phenotypic and functional enhancements in the hiPSC-HPCs over weeks of in vitro culture. Specifically, we find improved morphological organization, higher liver-specific gene expression levels, increased metabolic product secretion, and enhanced cytochrome P450 induction. The application of bioprinting technology in tissue engineering enables the development of a 3D biomimetic liver model that recapitulates the native liver module architecture and could be used for various applications such as early drug screening and disease modeling.3D bioprinting | in vitro hepatic model | iPSC | tissue engineering | biomaterials T he liver plays a critical role in the synthesis of important proteins and the metabolism of xenobiotic; the failure of these functions is closely related to disease development and drug-induced toxicity (1). For these reasons, in vitro liver models have been extensively developed to serve as platforms for pathophysiological studies and as alternatives to animal models in drug screening and hepatotoxicity prediction (2-4). Human primary hepatocytes, considered one of the most mature liver cell sources, lose many liver-specific functions rapidly when cultured in vitro due to the great discrepancies between the native and culture environments (5, 6). In addition, the practical difficulties in obtaining liver biopsy samples from every patient further hinder their use in personalized liver models. Consequently, hepatocytes derived from human induced pluripotent stem cells (hiPSCs), with the potential to be patient specific and easily accessible, have been widely acknowledged as the most promising cell source for developing personalized human hepatic models (4, 7).Many groups have reported monolayer differentiation of hiPSCs into hepatocyte-like cells (HLCs) and their ability to metabolize drugs (7-9). Nevertheless, hiPSC-derived HLCs are still considered immature in terms of many liver-specific gene expressions, functions, and...

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Section: Discussionsupporting

confidence: 84%

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confidence: 99%

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

Zhu

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

746

661

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“…To date, many researchers have reported the differentiation of human iPS cells into hepatocytes, which, like enterocytes, are important in drug pharmacokinetics (Si-Tayeb et al, 2010;Takayama et al, 2012;Ma et al, 2013). We have also established a method for the differentiation of human iPS cells into functional hepatocytes showing drug-metabolizing enzyme activities and inducibility (Kondo et al, 2014a). We found that histone deacetylase inhibitors promoted the hepatic differentiation of human iPS cells (Kondo et al, 2014b).…”

Section: Introductionmentioning

confidence: 89%

Generation of Enterocyte-Like Cells with Pharmacokinetic Functions from Human Induced Pluripotent Stem Cells Using Small-Molecule Compounds

et al. 2015

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The small intestine plays an important role in all aspects of pharmacokinetics, but there is no system for the comprehensive evaluation of small-intestinal pharmacokinetics, including drug metabolism and absorption. In this study, we aimed to construct an intestinal pharmacokinetics evaluation system and to generate pharmacokinetically functional enterocytes from human induced pluripotent stem cells. Using activin A and fibroblast growth factor 2, we differentiated these stem cells into intestinal stem cell-like cells, and the resulting cells were differentiated into enterocytes in a medium containing epidermal growth factor and small-molecule compounds. The differentiated cells expressed intestinal marker genes and drug transporters. The expression of sucrase-isomaltase, an intestine-specific marker, was markedly increased by small-molecule compounds. The cells exhibited activities of drug-metabolizing enzymes expressed in enterocytes, including CYP1A1/2, CYP2C9, CYP2C19, CYP2D6, CYP3A4/5, UGT, and sulfotransferase. Fluorescence-labeled dipeptide uptake into the cells was observed and was inhibited by ibuprofen, an inhibitor of the intestinal oligopeptide transporter solute carrier 15A1/PEPT1. CYP3A4 mRNA expression level was increased by these compounds and induced by the addition of 1a,25-dihydroxyvitamin D 3 . CYP3A4/5 activity was also induced by 1a,25-dihydroxyvitamin D 3 in cells differentiated in the presence of the compounds. All these results show that we have generated enterocyte-like cells that have pharmacokinetic functions, and we have identified small-molecule compounds that are effective for promoting intestinal differentiation and the gain of pharmacokinetic functions. Our enterocyte-like cells would be useful material for developing a novel evaluation system to predict human intestinal pharmacokinetics.

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“…Similar to Chen et al, Kondo and co-workers generated hepatocytelike cells by sequential stimulation with growth factors [89] . However, while Chen and co-workers utilized IMDM medium from day 8 to 12, the Kondo protocol requires the use of Modified Laford medium from day 12 to 21.…”

Section: Generation Of Hepatocytes From Ips Cells Using Growth Factorsmentioning

confidence: 87%

Induction of Hepatocyte Differentiation in Human Pluripotent Stem Cells

Tomizawa

Shinozaki

Motoyoshi

et al. 2015

Journal of Gastroenterology and Hepatology Research

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be advantageous in that it could resolve the ethical problems associated with stem cell research and could eliminate the need for administration of immunosuppresants in hepatocyte transplantation. To establish a method for the production of hepatocytes from hiPS cells, it is necessary to understand both the mechanism of hepatocyte differentiation as well as the current techniques for culturing of primary hepatocytes. Meanwhile, hepatocytes can also be differentiated from endodermal cells upon stimulation with growth factors that induce expression of specific transcriptional regulators. Current protocols for the generation of hepatocytes from hiPS cells include the sequential application of growth factors to mimic the environment of fetal liver. Initially, cells are treated with activin and bone morphogenetic proteins, followed by the application of hepatocyte growth factor and, at the final differentiation stage, Oncostatin M. Expression of transcription factors is necessary for hepatocyte differentiation. However, because the efficiency at which vectors expressing these factors can be transfected into hiPS cells is disappointingly low, adenoviral vectors have been used, which have a transduction efficiency of 100%. This method is used to sequentially introduce sex-determining region Y (Sry) HMG box (Sox) 17, hematopoietically expressed homeobox, and hepatocyte nuclear factor-4α into hiPS cells. These factors, in concert with the stimulation with growth factors, promote differentiation of these cells to hepatocytes. Despite the development of these procedures, however, the resulting cells remain immature and only perform certain hepatocyte functions. In this review, we describe each of the above points, and then discuss novel approaches and future directions for the In vitro production of hepatocytes. ABSTRACTCulturing of hepatocytes is used in both experimental and clinical procedures. Human induced pluripotent stem (hiPS) cells can be established from the somatic cells of individual patients. The In vitro production of hepatocytes from these hiPS cells would

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An Efficient Method for Differentiation of Human Induced Pluripotent Stem Cells into Hepatocyte-like Cells Retaining Drug Metabolizing Activity

Cited by 54 publications

References 41 publications

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

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

Generation of Enterocyte-Like Cells with Pharmacokinetic Functions from Human Induced Pluripotent Stem Cells Using Small-Molecule Compounds

Induction of Hepatocyte Differentiation in Human Pluripotent Stem Cells

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