isolation and expansion of human pluripotent stem cell derived hepatic progenitor cells by growth factor defined serum free culture conditions

Experimental Cell Research (xxxx) xxxx–xxxx Contents lists available at ScienceDirect Experimental Cell Research journal homepage: www.elsevier.com/locate/yexcr Isolation and expansion of human pluripotent stem cell-derived hepatic progenitor cells by growth factor defined serum-free culture conditions Takayuki Fukudaa, Kazuo Takayamab,c,d,e, Mitsuhi Hirataa, Yu-Jung Liua, Kana Yanagiharaa, ⁎ Mika Sugaa, Hiroyuki Mizuguchib,c,f,g, Miho K Furuea, a Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8, Saito-Asagi, Ibaraki, Osaka 567-0085, Japan Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 5650871, Japan c Laboratory of Hepatocyte Regulation, National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8, Saito-Asagi, Ibaraki, Osaka 567-0085, Japan d PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan e K-CONNEX, Kyoto University, Yoshida Honmachi, Sakyo-ku, Kyoto 606-8501, Japan f iPS Cell-based Research Project on Hepatic Toxicity and Metabolism, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan g Global Center for Medical Engineering and Informatics, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan b A R T I C L E I N F O A BS T RAC T Keywords: Hepatic progenitor cells Hepatic differentiation Hepatoblasts Hepatocytes Human pluripotent stem cells Serum-free culture Limited growth potential, narrow ranges of sources, and difference in variability and functions from batch to batch of primary hepatocytes cause a problem for predicting drug-induced hepatotoxicity during drug development Human pluripotent stem cell (hPSC)-derived hepatocyte-like cells in vitro are expected as a tool for predicting drug-induced hepatotoxicity Several studies have already reported efficient methods for differentiating hPSCs into hepatocyte-like cells, however its differentiation process is time-consuming, laborintensive, cost-intensive, and unstable In order to solve this problem, expansion culture for hPSC-derived hepatic progenitor cells, including hepatic stem cells and hepatoblasts which can self-renewal and differentiate into hepatocytes should be valuable as a source of hepatocytes However, the mechanisms of the expansion of hPSC-derived hepatic progenitor cells are not yet fully understood In this study, to isolate hPSC-derived hepatic progenitor cells, we tried to develop serum-free growth factor defined culture conditions using defined components Our culture conditions were able to isolate and grow hPSC-derived hepatic progenitor cells which could differentiate into hepatocyte-like cells through hepatoblast-like cells We have confirmed that the hepatocyte-like cells prepared by our methods were able to increase gene expression of cytochrome P450 enzymes upon encountering rifampicin, phenobarbital, or omeprazole The isolation and expansion of hPSCderived hepatic progenitor cells in defined culture conditions should have advantages in terms of detecting accurate effects of exogenous factors on hepatic lineage differentiation, understanding mechanisms underlying self-renewal ability of hepatic progenitor cells, and stably supplying functional hepatic cells Introduction Primary hepatocytes are used as a tool for predicting drug-induced hepatotoxicity during drug development However, their limited growth potential, narrow ranges of sources, and difference in variability and functions from batch to batch cause a problem Generation of homogenous hepatocyte-like cells derived from human pluripotent stem cell (hPSC) is expected as a solution Several studies have already reported efficient methods that differentiation of hPSCs into hepatocyte-like cells which exhibit hepatic gene expression and functions [1– Abbreviations: hPSC, human pluripotent stem cell; HPC, hepatic progenitor cell; FGF, fibroblast growth factor; BMP, bone morphogenetic protein; DE, definitive endoderm; hPSCHPC, hPSC-derived HPC; HPC-HBC, HPC-derived hepatoblast-like cell; HPC-HC, HPC-derived hepatocyte-like cell; hESC, human embryonic stem cell; iPSC, induced pluripotent stem cell; MEF, mouse embryo fibroblast; FAF-BSA, fatty acid–free bovine serum albumin; FOXA2, forkhead box A2; hPSC-HLPC, hPSC-derived hepatic lineage progenitor cell; HNF1α, hepatocyte nuclear factor alpha; HGF, hepatocyte growth factor; BIO, (2′Z,3′E)−6-Bromoindirubin-3′-oxime; EGF, epidermal growth factor; NIC, nicotinamide; DEX, dexamethasone; DAPT, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester; BDM, biliary differentiation medium; ICG, indocyanine green; HNF4α, hepatocyte nuclear factor alpha; AFP, alpha fetoprotein; GFX, GF109203X; ALB, albumin; CYP3A7, cytochrome P450 3A7; ITGB4, Integrinβ4 ⁎ Corresponding author E-mail address: mkfurue@nibiohn.go.jp (M.K Furue) http://dx.doi.org/10.1016/j.yexcr.2017.02.022 Received 20 July 2016; Received in revised form 14 February 2017; Accepted 15 February 2017 0014-4827/ © 2017 Published by Elsevier Inc Please cite this article as: Fukuda, T., Experimental Cell Research (2017), http://dx.doi.org/10.1016/j.yexcr.2017.02.022 Experimental Cell Research (xxxx) xxxx–xxxx T Fukuda et al (WO2012/104936) modified from hESF9 medium, which we previously developed for culturing hESCs [21] hESF-FX medium consisted of mESF basal medium (Wako Pure Chemical Industries, Osaka, Japan, http://www.wako-chem.co.jp/) supplemented with factors (10 µg/ml human recombinant insulin, µg/ml human apotransferrin, 10 µM 2-ethanolamine, 10 µM 2-mercaptoethanol, 20 nM sodium selenite), 9.4 µg/ml oleic acid conjugated with mg/ml recombinant human serum albumin (all from Sigma-Aldrich), mg/ml L-ascorbic acid phosphate (Wako Pure Chemical Industries), or 10 ng/ml FGF2, and ng/ml activin A (R & D Systems, Minneapolis, MN, http://www.rndsystems.com) The experiments using hESCs were performed following the Guidelines for utilization of hESCs of the Ministry of Education, Culture, Sports, Science and Technology of Japan with the approval by the institutional research ethics committee 5] Protocols by Takayama et al [6,7] employ transduction of adenoviral vector with transcription factor genes Since adenovirus vectors are one of the most efficient gene delivery vehicles that can provide high transduction efficiency in both dividing and non-dividing cells, differentiation into hepatocyte-like cells should be comparatively stable Nevertheless, differentiation efficiency into hepatocyte-like cells varies between experiments Moreover, the differentiation processes toward hepatocyte-like cells from hPSCs are time-consuming, laborintensive, and cost-intensive, as well as generated hepatocyte-like cells are inhomogeneous Thus, isolation and expansion of hepatic progenitor cells (HPCs) that have both ability of self-renewal and differentiation into functional hepatocytes would be ideal Reid's group [8–10] has proposed that there are types of HPCs which can give rise to liver: hepatic stem cells (HSCs) and hepatoblasts in fetal and adult liver Recent accumulated studies have demonstrated that HSCs can self-renew and differentiate into hepatoblasts which can differentiate into hepatocytes or bile ductal cells [9,11,12] During embryonic development, several kinds of hepatic lineage progenitor cells (HLPCs) are generated from ventral foregut endoderm [13] The ventral foregut endoderm stimulated by fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) gives rise to hepatic diverticulum The hepatic diverticulum cells expand to form liver bud and differentiate into hepatoblast, which differentiate into hepatocytes or bile ductal cells Usually, methods that promote hPSCs differentiation into hepatocyte-like cells in vitro are comprised of steps: differentiation of hPSCs into definitive endoderm (DE), induction into hepatic lineage, and maturation into hepatocyte-like cells These findings imply that several types of HLPC, including hepatic stem cells and hepatoblasts which can self-renewal and differentiate into hepatocytes, may exist and have a variety of property in different conditions Previous studies [14,15] reported that hPSC-derived hepatoblast-like cells can be maintained and expanded under serum containing culture conditions In current study, we developed growth factor defined serum-free culture conditions for growing hPSC-derived HPCs (hPSC-HPCs), and expanding HPC-derived hepatoblast-like cells (HPC-HBCs) which can differentiate toward hepatocyte-like cells (HPC-HCs) Isolation of hPSC-HPCs in our culture conditions should have advantages to detecting effects of exogenous factors on hepatic lineage differentiation, understanding underlying self-renewal ability of HPCs, and establishing stable supply of functional hepatic cells for pharmaceutical research 2.2 Culture of primary human hepatocytes Cryopreserved primary human hepatocytes (VERITAS, Tokyo, Japan, http://www.veritastk.co.jp/) were cultured according to the manufacturer's instructions Briefly, the hepatocytes were seeded at 25×105 cells/cm2 in hepatocyte culture medium (Lonza, Basel, Switzerland, http://www.lonza.com/) containing 10% FBS (Thermo Fisher Scientific) onto type I collagen (Nitta gelatin, Sendai, Japan, http://www.nitta-gelatin.co.jp/)-coated plates The medium was replaced h after seeding Experiments for drug response were conducted with the hepatocytes that were cultured for 48 h after plating the cells, as previously described [6,7] Cultures of primary human fetal hepatocytes, human normal fetal liver-CD34+ cells, and HepaRG® cells which are terminally differentiated hepatic cells derived from a human hepatic progenitor cell line are described in Supplementary information 2.3 Preparation of hPSC-derived hepatic lineage progenitor cells Hepatic lineage differentiation of Dotcom cells using adenovirus vectors was performed as described previously [6] Briefly, to promote mesendoderm differentiation, hPSCs were cultured for days on Matrigel (Corning, Corning, NY, https://www.corning.com/) in a differentiation medium consisting of hESF-DIF medium (Cell Science & Technology Institute, Sendai, Japan, http://cstimedia com/) supplemented with factors, 0.5 mg/ml fatty acid–free bovine serum albumin (FAF-BSA; Sigma-Aldrich), and 100 ng/ml activin A To generate DE-like cells, hPSC-derived mesendoderm cells were transduced with forkhead box A2 (FOXA2)-expressing adenovirus vectors on day and cultured until day on Matrigel in the differentiation medium To generate hPSC-HLPCs, the DE-like cells were transduced with FOXA2- and hepatocyte nuclear factor 1α (HNF1α)-expressing adenovirus vectors on day and cultured for days on Matrigel in hepatocyte culture medium (Lonza) supplemented with 30 ng/ml bone morphogenetic protein (BMP4; R & D Systems) and 20 ng/ml FGF4 (R & D Systems) Hepatic lineage differentiation of Tic and H9 cells was performed according to respective methods as described in Supplementary Information Materials and methods 2.1 hPSCs culture A human embryonic stem cell (hESC) line H9 (WA09) [16,17] was obtained from WISC Bank (WiCell Research Institute, Madison, WI, http://www.wicell.org/) Human lung fibroblast cell MRC-5 [18] -derived induced pluripotent stem cell (iPSC) lines, Tic (JCRB 1331), and Dotcom (JCRB 1327) [19,20] were obtained from the JCRB Cell Bank (National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan, http://cellbank.nibiohn.go.jp/) H9, Tic, and Dotcom cell lines were maintained on irradiated-inactivated mouse embryo fibroblast (MEF) feeder cells in KnockOut™ serum replacement (KSR, Thermo Fisher Scientific, Waltham, MS, https:// www.thermofisher.com/)-based medium supplemented with 10 ng/ml FGF2 (Katayama Chemical Industries, Osaka, Japan, http:// katayamakagaku.co.jp/) [16] KSR-based medium consisted of KnockOut™ DMEM/F-12 (Thermo Fisher Scientific) supplemented with 20% KSR, 0.1 mM 2-mercaptoethanol (Sigma-Aldrich, St Louis, MO, http://www.sigmaaldrich.com), mM L-glutamine (Thermo Fisher Scientific), 0.1 mM non-essential amino acids (Thermo Fisher Scientific), and or 10 ng/ml human recombinant FGF2 Prior to cell differentiation, H9 and Tic cells were maintained without feeder cells on µg/cm2 bovine fibronectin (Sigma-Aldrich) in hESF-FX 2.4 Development of culture conditions for hPSC-HPCs and HPCHBCs hPSC-HLPCs were seeded on plastic dish coated with bovine fibronectin (Sigma-Aldrich) at μg/cm2 in a serum-free medium for culturing HPCs, designated as HepSCF medium (Supplementary Table S1) HepSCF medium (JP2015-006137A) consists of HepSCF basal medium supplemented with factors (10 µg/ml human recombinant insulin, µg/ml human apo-transferrin, 10 µM 2-ethanolamine, 10 µM 2-mercaptoethanol, 25 nM sodium selenite), 100 ng/ml bovine heparan sulfate sodium salt (Sigma-Aldrich), and mg/ml FAF-BSA (Merck Experimental Cell Research (xxxx) xxxx–xxxx T Fukuda et al The cells were cultured with 20 ng/ml Wnt3a (R & D Systems) and/or 10 μM N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT; Sigma-Aldrich) to induce hepatocyte differentiation at 37 °C in 5% CO2 incubator The medium was changed every days (48-well plate) and days (24-well plate) for 30 days and 24 days respectively For cholangiocyte differentiation, hPSC-HBCs were cultured in three-dimensional collagen gel Premixture gel solution was prepared with solubilized type I collagen in HCl (pH 3.0), 10x concentrated DMEM medium, and solution of 50 mM NaOH containing 200 mM HEPES and 2.2% NaHCO3 at mixing ratio of 8: 1: and gently mixed on ice Next, hPSC-HBCs were harvested with 0.1% dispase and suspended in biliary differentiation medium (BDM) BDM consisted of DMEM/F12 medium supplemented with factors, mg/ml FAFBSA, 10 mM NIC, 0.1 µM DEX, 20 ng/ml HGF, and 20 ng/ml EGF The cell suspension in BDM was mixed with the premixture gel solution at the volume ratio of 2:1 (a final concentration of type I collagen: 0.8 mg/ml) Cells suspended in the collagen gel solution were seeded in aliquots of 0.25 ml into each well of a 24-well plastic plate and incubated at 37 °C for 30 in CO2 incubator to be solidified Then, ml of BDM was overlaid on the solidified gel Next day, the medium was replaced by a fresh BDM supplemented with 1% FBS (Equitech-Bio, Kerrville, TX, http://www.equitech-bio.com/), and the cells were cultured for 10 days with every days medium replacement Millipore) Effects of several growth factors including hepatocyte growth factor (HGF), BMP4, and FGF7 (all from R & D Systems) and several small molecules including Y27632 (Wako Pure Chemical Industries), SB431542 (Tocris Bioscience, Bristol, UK, http://www tocris.com/), LY294002 (Cell Signaling Technology, Danvers, MA, http://www.cellsignal.com/), GF109203X (GFX; Tocris Bioscience), U0126 (Promega, Madison, WI, www.promega.com), (2′Z,3′E)−6Bromoindirubin-3′-oxime (BIO; Wako Pure Chemical Industries), epidermal growth factor (EGF; R & D Systems), nicotinamide (NIC; Sigma-Aldrich), dexamethasone (DEX; Sigma-Aldrich) were examined on the culture of hPSC-HPCs and HPC-HBCs The cells were passaged using EZPassage™ tool (Thermo Fisher Scientific) or 0.1% dispase (Roche Diagnostics, Basel, Switzerland, http://www.roche-appliedscience.com) before becoming confluent Sprit ratio was 1:3–1:4 Culture medium was changed every days 2.5 Assessment of cell viability The Alamar Blue assay was used to assess cell viability The cells were seeded on 96-well plates at a cell density of 1×104 cells and cultured for days with medium replacement on culture day On culture day 7, AlamarBlue® reagent (Thermo Fisher Scientific) was added into each test well and after three-hour incubation in CO2 incubator at 37 °C, the fluorescence intensity in each well was measured by a microplate reader (EnSpire PerkinElmer, Waltham, MA, http://www.perkinelmer.com/) 2.9 Uptake and release of indocyanine green The hPSC-HPC-derived hepatocyte-like cells (HPC-HCs) were incubated with mg/ml indocyanine green (ICG; Sigma-Aldrich) in HepSCF medium supplemented with 20 ng/ml HGF, 10 μM SB431542, 10 μM Y27632, µM DEX, 10 mM NIC, 0.1 mg/ml Lascorbic acid phosphate, 20 ng/ml oncostatin-M, 20 ng/ml Wnt3a, and 10 μM DAPT at 37 °C for h After washing times with phosphatebuffered saline, the cells were incubated in fresh medium without ICG at 37 °C for h Images of cellular uptake and release of ICG were captured by microscopy (M125 Leica, Wetzlar, Germany, http://www leica-microsystems.com/) 2.6 Immunohistochemistry Immunohistochemistry was performed as described previously [22] Briefly, fixed cells were incubated with primary antibody (Supplementary Table S2) at °C for overnight followed by incubation with secondary antibody at room temperature for h Nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific) Images were taken with a fluorescence microscope (Biozero BZ-9000 KEYENCE, Osaka, Japan, http://www.keyence.co.jp/) or an image analyzer (In Cell Analyzer 2000; GE Healthcare Life Sciences, Little Chalfont, UK http://www.gelifesciences.com/) 2.10 CYP induction test 2.7 RNA isolation and quantitative real-time PCR HPC-HCs and adult hepatocytes were treated with 20 mM rifampicin, 500 mM phenobarbital at 37 °C for 48 h, or 50 mM omeprazole (all from Sigma-Aldrich) at 37 °C for 24 h, as previously described [6,7] Dimethyl sulfoxide was used as negative control RNA extracted from the cells was analyzed as described above Total RNA was purified from the cells using Micro-RNeasy kit (Qiagen, Valencia, CA, http://www.qiagen.com/) cDNA synthesis was performed using Superscript VILO cDNA synthesis kit (Thermo Fisher Scientific) Quantitative real-time PCR (qRT-PCR) was performed with SYBR® Premix Ex Taq™ II (TaKaRa, Shiga, Japan, http://www.takarabio.co.jp/) on Applied Biosystems 7300 Real-Time PCR system (Thermo Fisher Scientific) The primer sequences are shown in Supplementary Table S3 One-step reverse transcription and realtime PCR from total RNA was performed using Cells-to-CT ™ 1-Step TaqMan® Kit with TaqMan® Gene Expression Assays (Thermo Fisher Scientific) Primer-probe sets are shown in Supplementary Table S4 Results 3.1 Derivation of hPSC-HPCs from hPSC-HLPCs To develop growth factor defined serum-free culture condition for serially culturing HLPCs, effects of several growth factors on the cell growth were examined hPSC-HLPCs prepared as described in the material and methods were seeded at the cell density of 2–4×105 cells /well on 6-well plates coated with µg/cm2 bovine fibronectin in HepSCF medium supplemented with BMP4, HGF, or FGF7 Within passages, tightly packed small colonies appeared in any of the conditions tested and consisted of smaller cells with higher nucleus-tocytoplasm ratio compared with hepatoblast-like cells (Supplementary Fig S1A) However, the tightly packed colonies began to disappear around passage when the cells were cultured in the medium supplemented with BMP4, BMP4+HGF, BMP4+FGF7, or without any growth factors In the culture supplemented with HGF, FGF7, HGF+FGF7 or BMP4+HGF+FGF7, cells formed larger tightly packed colonies during passages To characterize hPSC-HLPCs grown in the above culture condi- 2.8 Differentiation into hepatocytes or cholangiocytes from hPSCHBCs HPC-HBCs were cultured with HepSCF medium supplemented with 10 ng/ml HGF, 10 ng/ml FGF7, 10 μM SB431542, 10 μM Y27632, 10 mM NIC, and 0.1 µM DEX (designated as HepSCF-6F medium) for several passages, preceding differentiation experiments For hepatocyte differentiation, hPSC-HBCs were inoculated on a 48well plate or 24-well plate coated with bovine fibronectin at µg/cm2 in HepSCF-6F medium Upon reaching confluent density, medium was changed to HepSCF medium supplemented with 20 ng/ml HGF, 10 μM SB431542, 10 μM Y27632, µM DEX, 10 mM NIC, 0.1 mg/ml Lascorbic acid phosphate, and 20 ng/ml oncostatin-M (R & D Systems) Experimental Cell Research (xxxx) xxxx–xxxx T Fukuda et al Fig Proliferation of hPSC-HLPCs cultured in HepSCF medium supplemented with growth factor(s) (A): hPSC-HLPCs (derived from Dotcom cells) cultured in HepSCF medium supplemented with HGF and FGF7 at fifth passage were subjected to immunostaining with antibodies of hepatic lineage markers, anti-FOXA2 (green), atni-HNF4α (blue), and antiAFP (red) The bar represents 100 µm (B): Graphic representation of ratio of hPSC-HLPCs (derived from Dotcom cells) expressing FOXA2, HNF4α, or AFP at fifth passage calculated by image analyzer Data are represented as means ± SD (n=3) (C): Gene expressions of hPSC-HPCs (derived from H9 cells) cultured in HepSCF-2F medium were examined by qRT-PCR The gene expression levels were compared with undifferentiated cells and normalized against that of hPSC-HPCs cultured in HepSCF-2F medium Data are represented as means ± SD (n=4 or 6) (D): Immunostaining images of hPSC-HPCs (derived from H9 cells) of tightly packed colony selected from hPSC-HLPCs cultured in HepSCF-2F medium Cells were stained with antibodies of hepatic stem markers and hepatoblast markers (green or red) Nuclei were counterstained with Hoechst 33342 (blue) The bar represents 50 µm Abbreviations: BF, bright field; GF, growth factor; undif, undifferentiated hPSCs; F, hPSC-HPCs cultured in HepSCF-2F medium **, P < 0.01; ***, P < 0.005 amount of cytoplasm They also reported that HSCs not express AFP whereas hepatoblasts express AFP [9] These findings suggest that the cells of tightly packed colonies which were less stained with anti-AFP antibodies are HSC-like cells Collectively, these data indicated that HepSCF medium supplemented with FGF7+HGF (designated as HepSCF-2F medium) supported culture of HSC-like cells However, morphological heterogeneity was observed among the cells, thus tightly packed colonies were manually isolated as HSC-like cells and grown in HepSCF-2F medium The harvested cells adhered, proliferated slowly, and formed tightly packed colonies on a 6-well plate coated with fibronectin (Supplementary Fig S1C) Quantitative RT-PCR (qRT-PCR) analysis confirmed the expression levels of hepatic lineage progenitor markers were higher in HSC-like cells than in undifferentiated hPSCs (Fig 1C, Supplementary Fig S1D) To characterize the cells grown in these culture conditions, the cells were immunostained by antibodies for hepatic lineage marker genes The cells were strongly immunopositive for NCAM, CD29, KLF5, SOX17 CK19, FOXA2, HNF4α, SOX9, tions, expression of endoderm or hepatic lineage markers was examined by immunohistochemistry The cells cultured for 5–8 days after one to five passages were stained with antibodies for an endoderm marker FOXA2, an early hepatoblast marker hepatocyte nuclear factor alpha (HNF4α), or a late hepatoblast marker alpha fetoprotein (AFP) The cells cultured with BMP4, BMP4+HGF, BMP4+FGF7, or BMP4+HGF+FGF7, which were comparably flat and large, were intensely stained with AFP but much less stained with FOXA2 and HNF4α (Supplementary Fig S1B) The cells of tightly packed colonies cultured with HGF, FGF7, or HGF+FGF7 were intensely stained with FOXA2 and HNF4α but much less stained with AFP at third to fifth passage (Fig 1A, Supplementary Fig S1B) Analysis by the image analyzer showed that FOXA2 and/or HNF4α-positive cell ratio was higher in the cells cultured with FGF7+HGF, compared with those in other culture conditions at fifth passage (Fig 1B) Reid's group [9] reported that HSCs are comparatively small with a high nucleus-tocytoplasm ratio whereas hepatoblasts are flatter and larger with higher Experimental Cell Research (xxxx) xxxx–xxxx T Fukuda et al Fig (continued) To characterize the cells grown in these culture conditions, expression of hepatic lineage marker genes was examined by qRT-PCR The cells cultured with LY294002, GFX, and BIO did not grow, thus the cells cultured with Y27632, SB431542, or U0126 were analyzed for gene expression of FOXA2, HNF4α, NCAM, CK19, CD133, CD13, EpCAM, AFP, and Albumin (ALB) (Fig 2B) SB431542 decreased the expressions of HNF4α, NCAM, and CD133 and intensely increased those of AFP and ALB Y27632 decreased the expressions of FOXA2, HNF4α, NCAM, CD13, and EpCAM U0126 decreased the expressions of FOXA2, CK19, and CD133 and increased those of AFP and ALB Since Y27632 increased the cell viability, combined effect of Y27632 and SB431542 to hPSC-HPCs proliferation was examined in HepSCF-2F medium SB431542 increased the cell viability in a concentration dependent manner of Y27632 (Fig 2C) Addition of 10 µM SB431542 with 10 µM Y27632 increased the cell viability by 2.7 times and appeared alterative cell morphology, giving a cobblestonelike appearance (Supplementary Fig S2B, right third of the image) Compared to the cells cultured in HepSCF-2F medium, the cell size and cytoplasmic volume were increased Previous study has demonstrated that hepatoblasts derived from HSCs have morphology of larger in size with lower nucleus-to-cytoplasm ratio than HSCs [9] These findings suggest that the cells cultured in HepSCF-2F medium supplemented with 10 µM SB431542 and 10 µM Y27632 were hepatoblast-like cells To characterize the cells cultured under the influence of 10 µM SB431542 in combination with Y27632 (0, 1, 2, 5, 10 µM), the and EpCAM, and moderately for Dlk1 and ICAM AFP-positive cells were rarely detectable In contrast, CD13, ALB, and CK7 positive cells were hardly observed (Fig 1D, Supplementary Fig S1E-a) To examine whether HpSCF-2F medium is suitable to culture HSClike cells prepared by other differentiation protocols for hPSC-HLPCs, protocols reported by Vallier's group [3,23] or Duncan's group [4,24] were challenged with hiPSC line Tic and hESC line H9 Tightly packed colonies appeared and the cells intensely expressed FOXA2 and slightly expressed AFP (Supplementary Fig S1E-b, c) hPSCs-derived HSC-like cells are referred as hPSC-derived HPCs (hPSC-HPCs) 3.2 Development of culture conditions for HPC-derived hepatoblastlike cells proliferation Although hPSC-HPCs were successively cultured in HepSCF-2F medium, cell growth seemed very slow To modify HepSCF-2F medium, effect of small molecules LY294002, GFX, SB431542, U0126, BIO, and Y27632 on cell proliferation of hPSC-HPCs was examined Cell viability assay using Alamar Blue showed that addition of Y27632 almost doubled the viability of cells cultured with HepSCF2F (Fig 2A) The addition of SB431542 or U0126 hardly changed the viability of cells, while LY294002 decreased the viability In the presence of GFX or BIO, the cell viability was extremely low and eventually the cells were entirely extinguished in days (Fig 2A, Supplementary Fig S2A) Experimental Cell Research (xxxx) xxxx–xxxx T Fukuda et al Fig (continued) demonstrated that HepSCF-2F medium supplemented with 10 µM Y27632 and 10 µM SB431542 (designated as HepSCF-4F medium) supported the phenotype of HPC-derived hepatoblast-like cells (HPCHBCs) and promoted their proliferation Previous studies have reported that NIC and EGF enhance cell proliferation of hepatocytes isolated from rat liver without loss of hepatocyte-specific functions [27,28] DEX induces expression of both HNF4α and C/EBPα, which are essential to liver development [29] To examine the effect of these factors on HPC-HBCs, cell viability and gene expressions of the cells grown in HepSCF-4F medium supplemented with NIC+DEX or EGF were examined However, cell viability was not changed by NIC+DEX while EGF or EGF+NIC+DEX increased the viability (Supplementary Fig S3A) Addition of NIC+DEX hardly affected the expression of hepatic lineage genes while that of EGF decreased (Supplementary Fig S3B, S3C) Immunohistochemical study revealed that the cells cultured in HepSCF-4F supplemented with NIC +DEX expressed hepatic lineage markers ICAM, EpCAM, CD133, CD13, AFP, FOXA2, and ALB at high level, and NCAM at low level (Supplementary Fig S3D) These results indicated that the NIC+DEX maintained the cell phenotype of hepatoblast-like cells, however, EGF decreased expression of hepatic lineage genes was examined by qRT-PCR (Supplementary Fig S2C) The expression of FOXA2 was comparably increased by any of the supplementation condition The expressions of ICAM, AFP, and ALB were increased by SB431542 and Y27632 in a concentration dependent manner CK19 expression was increased by SB431542 and Y27632 in a concentration dependent manner, but cosupplementation of 10 µM SB431542 and 10 µM Y27632 did not affect the expression level The expressions of HNF4α, NCAM, CD13, CD133, and EpCAM were decreased by 10 µM SB431542, but recovered with co-supplementation of Y27632 in a concentration dependent manner Immunohistochemistry showed that these cells moderately expressed ICAM, CD133, and CD13, but NCAM at lower level (Supplementary Fig S2D) To characterize the cells cultured with 10 µM SB431542 and 10 µM Y27632, the expression of hepatic lineage genes was examined by qRTPCR (Fig 2D) and immunochemical study (Fig 2E) Compared with the gene expression in the cells cultured HepSCF-2F, the gene expression of NCAM, KLF5, SOX17, CK19, HNF4α, SOX9, SHH, LGR5, and DLK1 were decreased The expression of CD29, FOXA2, ICAM, and CD133, were not changed The expression of CD13, AFP, and ALB were increased Immunohistochemical examination showed that NCAM, CD29, KLF5, SOX17, and CK19 were not detected, while the cells were moderately immunopositive for FOXA2, HNF4α, and SOX9, and strongly immunopositive for EpCAM, DLK1, ICAM, CD13, AFP, and ALB Previous studies have reported that the expression of hepatoblasts markers, ICAM, AFP, and ALB, were high in hepatoblasts compared with HSCs [8,9,25] Differentiated HepaRG® cells which are derived from undifferentiated hepatic progenitor HepaRG® cells with morphological and functional characteristics of early hepatoblasts [26] were weakly or partially immunopositive for CD29, KLF5, CK19, ICAM, CD13, AFP, and ALB (Supplementary Fig S2E) Primary human fetal hepatocytes were weakly immunopositive for CD29, KLF5, SOX17, CK19, FOXA2, HNF4α, ICAM, CD13, and AFP, and strongly immunopositive for ALB (Supplementary Fig S2F) In the culture of fetal liver-CD34+ cells, which are considered as hepatic progenitor cells, the immunopositive cells for CD29, KLF5, CK19, ICAM, CD13, and AFP were observed (Supplementary Fig S2G) Overall, the results 3.3 Differentiation of HPC-HBCs into cholangiocyte-like and hepatocyte-like cells HSCs and hepatoblasts are known to differentiate into both cholangiocytes and hepatocytes To examine the differentiation potential of HPC-HBCs, cell differentiation into cholangiocytes and hepatocytes were tested To examine the differentiation activity of HPC-HBCs into cholangiocytes, the cells were cultured in three-dimensional culture conditions After the cells were embedded in 0.08% type I collagen gel, the cells were cultured in BDM supplemented with 1% FBS The cells branched out from the edge of aggregates and elongated gradually in 10-day culture (Fig 3A) On day 10, the cells were fixed or total RNA were extracted Whole mount immunochemical staining of the cells in collagen gel showed CK19 expression (Fig 3B) Analysis by qRT-PCR Experimental Cell Research (xxxx) xxxx–xxxx T Fukuda et al Fig Proliferation of hPSC-HPCs cultured in HepSCF-2F medium supplemented with small molecule(s) (A): Viability of hPSC-HPCs (derived from Dotcom cells) cultured in HepSCF-2F medium supplemented with small molecule(s) was calculated by Alamar Blue assay Data are represented as means ± SD (n=3) (B): Gene expressions of hPSC-HPCs (derived from Dotcom cells) cultured in HepSCF-2F medium supplemented with small molecule(s) were examined by qRT-PCR The gene expression levels were normalized against that of hPSC-HPCs cultured in HepSCF-2F medium without small molecules Data are represented as means ± SD (n=3) (C): Viability of hPSC-HPCs (derived from Dotcom cells) cultured in HepSCF-2F medium supplemented with SB431542 and/or Y27632 was calculated by Alamar Blue assay Data are represented as means ± SD (n=3) (D): Gene expressions of hPSCHPCs (derived from H9 cells) cultured in HepSCF-2F medium supplemented with SB431542 and Y27632 were examined by qRT-PCR The gene expression levels were normalized against that of hPSC-HPCs cultured in HepSCF-2F medium without SB431542 and Y27632 Data are represented as means ± SD (n=4, or 6) (E): hPSC-HPCs (derived from H9 cells) cultured in HepSCF-2F medium supplemented with SB431542 and Y27632 were subjected to immunostaining with antibodies of hepatic stem markers and hepatoblast markers (green or red) Nuclei were counterstained with Hoechst 33342 (blue) The bar represents 50 µm Abbreviations: untreat, untreatment; SB, SB431542; Y, Y27632; GFX, GF109203X; F, hPSC-HPCs cultured in HepSCF-2F medium *, P < 0.05; **, P < 0.01; ***, P < 0.005 Experimental Cell Research (xxxx) xxxx–xxxx T Fukuda et al Fig (continued) Fig (continued) Experimental Cell Research (xxxx) xxxx–xxxx T Fukuda et al Fig Differentiation of HPC-HBCs (derived from Dotcom cells) into cholangiocyte-like cells in collagen gel 3D-culture (A): HPC-HBCs differentiated into cholangiocyte-like cells in collagen gel at Day (left) and Day 10 (right) The bar represents 100 µm (B): Cholangiocyte-like cells were subjected to immunostaining with anti-CK19 antibody The bar represents 100 µm (C): The gene expressions of cholangiocyte-like cells were examined by qRT-PCR The gene expression levels were normalized against that of HPC-HBCs cultured in HepSCF-4F medium All data are represented as means ± SD (n=3) Abbreviations: undif, undifferentiated hPSCs; ChCs, cholangiocyte-like cells **, P < 0.01; ***, P < 0.005 3.4 Functions of HPC-HBC-derived hepatocyte-like cells also revealed that CK19, Integrinβ4 (ITGB4), Aquaporin-1 (AQP1), and Secretin receptor (SCTR) were overexpressed compared with that of HPC-HBCs (Fig 3C) To examine the differentiation activity of HPC-HBCs into hepatocytes, cells were subjected to several differentiation protocols for hepatocytes described previously [2–4] However, treatment with these protocols resulted in cell death or changed morphology into which was not of hepatocytes Then, we modified HepSCF-6F medium (6F: FGF7, HGF, SB431542, Y27632, NIC, and DEX) for differentiation toward hepatocytes to HepSCF-7F medium (7F: HGF, SB431542, Y27632, NIC, DEX, L-ascorbic acid-2-phophate, and oncostatin-M) and additional effect of DAPT (HepSCF-7F+DAPT) and/or Wnt3a (HepSCF-7F +DAPT+Wnt3a) were tested HPC-HBCs were cultured on fibronectin in HepSCF-7F, HepSCF-7F+DAPT, or HepSCF-7F+DAPT+Wnt3a for 16 and 24 days The expression levels of most of hepatic marker genes and Cytochrome P450 (CYP) enzymes were higher in the cells cultured for 24 days than for 16 days (Supplementary Fig S4A) As compared with undifferentiated hPSCs or HPC-HBCs cultured in HepSCF-4F medium, the expressions of hepatocyte markers (ALB, AAT, UGT1A1, TDO, GSTA1, and ASGR1), and CYP enzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, and CYP7A1) in the hPSC-HPCs cultured in HepSCF-7F, HepSCF-7F +DAPT, or HepSCF-7F+DAPT+Wnt3a were higher than those in the HPC-HBCs, while those of AFP, TTR and CYP1A1 were lower (Fig 4A and Supplementary Fig S4A) Furthermore, the expressions of hepatocyte markers and CYP enzymes in the HPC-HBCs cultured in HepSCF-7F+DAPT+Wnt3a were highest among those conditions The expressions of these marker genes in other cell line also showed similar results (Supplementary Fig S4B) Immunohistochemistry showed that the cells cultured in HepSCF-7F+DAPT+Wnt3a were intensely stained with anti-CYP3A4, CK18, AAT, or ALB antibodies, whose positive cell ratio were 87.6%, 95.4%, 94.6%, and 91.8% respectively (Fig 4B) Hepatocytes are known to have important function to detoxify both exo- and endogenous chemical compounds Functional quality of HPCHBC-derived hepatocyte-like cells (HPC-HCs) was examined by uptake and excretion of ICG and drug-induced CYP gene expression ICG uptake and excretion were confirmed in HPC-HCs which were incubated in HepSCF-7F medium supplemented with ICG for 60 at 37 °C, followed by incubation in fresh HepSCF-7F medium for h at 37 °C (Fig 5A) Next, the CYP induction of HPC-HCs was examined Primary hepatocytes were adapted for culture when the cells were treated with drugs or chemicals The method was used as references of the FDA guidance for drug interaction studies [30], consensus model of Pharmaceutical Research and Manufacturers of America (PhRMA) member companies [31], or OECD Guidelines for the testing of chemicals [32], as previously described [6,7] Gene expression of CYPs was determined in HPC-HCs and cultured primary adult hepatocytes after drug treatment with omeprazole for CYP1A2, rifampicin for CYP3A4, or phenobarbital for CYP2B6, individually As a reference, each CYP gene expression was individually determined in primary hepatocytes freshly thawed which were used for each experiment The expressions of CYP1A2 by omeprazole and CYP3A4 by rifampicin in HPC-HCs were comparatively increased, as primary hepatocytes The expression of CYP2B6 in HPC-HCs by phenobarbital was significantly increased (Fig 5B) These results confirmed that HPC-HCs can function as hepatocyte-like cells Discussion The main purposes of this study were to develop serum-free defined culture conditions for hPSC-HPCs or HPC-HBCs, and to characterize Experimental Cell Research (xxxx) xxxx–xxxx T Fukuda et al Fig Differentiation of HPC-HBCs into HPC-HCs (A): Gene expressions of HPC-HCs (derived from H9 cells) were examined by qRT-PCR The gene expression levels were normalized against that of HPC-HBCs cultured in HepSCF-4F medium All data are represented as means ± SD (n=4 or 6) (B): HPC-HCs (derived from Dotcom cells) were subjected to immunostaining with anti-CYP3A4, anti-CK18, anti-AAT, and anti-ALB antibodies The bar represents 50 µm Abbreviations: undif, undifferentiated hPSCs; HBC, HPC-HBCs; 7F+Da, 7F+DAPT; 7F+Da+Wn, 7F+DAPT+Wnt3a *, P < 0.05; **, P < 0.01; ***, P < 0.005 that the human liver multipotent progenitor cells (hFLMPCs) isolated from human fetal liver are a small blast-like cells with a high nuclear to cytoplasm ratio The hFLMPCs express CD34, CD90, EpCAM, and CK19 but neither AFP nor ALB, and differentiate into hepatocyte-like and cholangiocyte-like cells Goldman et al [41] suggested that Kinase insert domain receptor (KDR) and CD31 can characterize different hepatic lineage cells Reid's group reported that HSCs express NCAM, Sonic Hedgehog, and CK19, while hepatoblasts express ICAM, AFP, and ALB [8–10] Both cells also express EpCAM and CD133 These results suggested that addition of SB431542 and Y27632 might support hepatoblast-like phenotype These findings demonstrate that several types of HLPCs may exist and have a variety of property in different conditions Culture conditions determined in this study were able to culture types of hPSC-HLPCs characterized as HSC-like cells and hepatoblast-like cells in HepSCF-2F or HepSCF-4F medium respectively Previously reported differentiation protocols for hPSCs toward hepatocytes use FBS, B27 supplement [42], or Matrigel, which contain undefined concentrations or components from raw materials [43] Because these materials have different quality from batch to batch, many batches have to be checked to obtain suitable one Our culture conditions for hPSC-HPCs, HPC-HBCs, and HPC-HCs consist of HepSCF medium supplemented with several factors (Supplementary expandable cells Here, we have developed growth factor defined serum-free culture conditions without feeder cells for isolating hPSCHPCs, proliferating HPC-HBCs, and promoting differentiation of HPCHBCs into HPC-HCs Because all the components of culture medium are defined, our culture conditions can constantly reproduce HPC-HCs We have confirmed that our culture condition for proliferation of HPCHBCs is capable of maintaining HPC-HBCs for at least months Our study showed that hPSC-HPCs cultured in HepSC-2F medium grow slowly and have comparatively small in size and tightly packed characteristics On the other hand, HPCs-HBCs cultured in HepSC-4F medium were comparatively flat and large in shape with high amount of cytoplasm hPSC-HPCs cultured in HepSC-2F medium expressed NCAM, CD29, KLF5, SOX17, CK19, HNF4a, SOX9, and EpCAM at higher levels, and ICAM, CD13, AFP, and ALB at lower levels, compared with HPC-HBCs grown in HepSC-4F Many reports of a variety of hepatoblast markers had been made such as EpCAM [25,33], DLK-1 [34], CD13 [35], CD133 [33,36], Lgr5 [37], and Carboxypeptidase M [15], concrete definition of hepatoblast is not yet identified Several studies have demonstrated that there are several stages of liver stem cells which have the capacity for unlimited proliferation and mutilineage differentiation, and such kind of liver stem cells change their expression markers in dependence of differentiation stages, location, or situation [38,39] Dan et al [40] reported 10 Experimental Cell Research (xxxx) xxxx–xxxx T Fukuda et al Fig (continued) Fig Evaluation of functions of HPC-HCs (A): HPC-HCs (derived from Dotcom cells) were examined for their ability of uptake (left) and release (right) for ICG The bar represents 100 µm (B): Induction of CYP450 enzymes by drugs was examined in HPC-HCs (derived from Dotcom cells), and primary human hepatocytes which were thawed and cultured for 48 h (PHH 48) The cells were treated with omeprazole for CYP1A2, rifampicin for CYP3A4, or phenobarbital for CYP2B6, individually (black bars) Each gene expression in primary human hepatocytes freshly thawed (PHH 0) was also examined individually (shaded bars) The gene expression levels were normalized by that of HPC-HCs with DMSO treatment as control cells (white bars) All data are represented as means ± SD (n=3) **, P < 0.01 11 Experimental Cell Research (xxxx) xxxx–xxxx T Fukuda et al (AMED) to MKF, and JSPS KAKENHI Grant Number JP16H05535 to MKF, MS, and TF and Japan Agency for Medical Research and Development (AMED) Grant Number 15bk0104005h0003 to HM Table S5) The HepSCF medium consists of HepSCF basal medium supplemented with factors, bovine heparan sulfate sodium salt, and FAF-BSA The factors have been used for culturing rat salivary gland stem cells [44,45], mouse ESCs [46], mouse neural crest cells [47], hESCs/hiPSCs [22,48], and human mesenchymal stem cells [49] under serum- and feeder-free conditions in our previous studies Several supplements including FGF7, HGF, SB431542, Y27632, NIC, and DEX supported the proliferation and phenotype of hPSC-HPCs or HPCHBCs FGF7 is not found in normal or developing liver cells but expressed during liver regeneration after toxin-induced hepatic injury [50] Expression of a FGF7 receptor, FGFR2, also increases in hepatocytes after toxin-induced hepatic injury [51] Gene targeting experiments showed that FGF7-/- mice is born normally, but exhibited severely suppression of cell proliferation in liver regeneration [50] The dominant-negative form of FGFR2b also exhibited reduction of cell proliferation after partial hepatectomy in adult mouse [52] HGF is well known to be required for hepatoblast proliferation [29,53–55] In the presence of FGF7 and HGF, a MEK inhibitor U0126 or a PI3K inhibitor LY294002 suppressed the proliferation of hPSC-HPCs These evidences indicate that both MAPK/Erk and PI3K signaling cascades triggered by FGF7 and/or HGF are indispensable for maintaining hPSC-HPCs culture TGF-β signaling regulates the differentiation of hepatoblasts toward bile ductal cells in liver development and regeneration [56–59] SB431542, a specific inhibitor of TGF-β receptors, ALK4, ALK5, and ALK7 were expected to prevent hPSC-HPCs from spontaneously differentiation into cholangiocytes In fact, SB431542 increased expression of hepatoblast markers, AFP and ALB These findings suggest that inhibition of TGF-β signaling may be advantageous to maintain phenotype of hepatoblast by inhibiting cell differentiation toward cholangiocytes Due to slow cell growth of hPSC-HPCs, it was difficult to perform many experiments and even to calculate cell growth rate A Rho kinase inhibitor Y27632 increased the viability of hPSC-HPCs in HepSCF-2F medium Y27632 protects hESCs from apoptosis [60], and promotes proliferation of several types of cells, such as limbal epithelial cells [61] These findings suggest that Rho signaling may be involved in survival or proliferation of HPCs or hepatoblasts during liver development or homeostasis We tested whether HPC-HBCs grown in HepSCF-4F or HepSCF-6F can be cryopreserved After a month of cryopreservation in liquid nitrogen, thawed cell exhibited a similar growth and morphology to those before freezing (Supplementary Fig S5A, S5B) Appendix A Supplementary material Supplementary data associated with this 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