EC and MC isolation and culture

Under the permission of the Ethical Review Board of Yokohama City University, human UCs were obtained from the Department of Obstetrics and Gynecology, Yokohama City University Hospital from full-term caesarian section births after obtaining informed consent from the mother. ECs and MCs were collected from the same UC. The collection procedure was in accordance with the ethical standards of the local ethics committee. To isolate ECs [17], the cord was laid out on a clean disk; the cord vein was washed three times with phosphate-buffered saline (PBS) and filled with 10 ml collagenase (1 mg/ml; Sigma-Aldrich, St. Louis, MO, USA). After clamping of the open end, the cord was incubated in RPMI 1640 medium at 37 °C for 15 min. The vein was washed with RPMI 1640 medium, the wash medium was collected and centrifuged at 200 × g for 5 min, and the pellet was resuspended in 10 ml EC growth medium (EGM; Lonza, Walkersville, MD, USA) and seeded in a 10-cm 0.1% gelatin-coated dish. For MC isolation, the vein and arteries were removed from the cord after isolation of ECs, and the cord was cut into pieces 1–2 mm3 that were incubated in an enzyme cocktail that included 2500 U/ml collagenase (Gibco, Grand Island, NY, USA), 5 mg/ml hyaluronidase (Wako Pure Chemical Industries, Osaka, Japan), and 2 U/ml dispase (Roche Diagnostics, Indianapolis, IN, USA) for 4 h with light shaking at 37 °C. After incubation, the sample was centrifuged at 400 × g for 10 min; the pellet was washed once with RPMI 1640 and resuspended in 10 ml mesenchymal stem cell growth medium (MSCGM; Lonza), and cells were seeded in a 10-cm tissue culture dish. ECs and BM-derived mesenchymal stem cells were obtained from Lonza as control ECs (con-ECs) and MCs (con-MCs) and maintained in EGM and MSCGM, respectively. All cells were maintained at 37 °C in a humidified incubator with 5% CO2.

Generation of nonviral feeder-free hiPSCs from UC-derived ECs

Feeder-free hiPSCs were reprogrammed from ECs using a protocol reported previously [18], with minor modifications. Briefly, ECs were transfected with episomal iPSC reprogramming vectors (pCXLE-hOCT3/4-shp53-F, pCXLE-hSK, pCXLE-hUL, and pCXWB-EBNA1) using Nucleofector 4D and then cultured in a plate coated with growth factor-reduced Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) in mTeSR medium (Stem Cell Technologies, Vancouver, BC, Canada). When the size of hiPSC colonies exceeded 1 mm, the colonies were picked and cultured in a plate coated with growth factor-reduced Matrigel in mTeSR medium to establish individual hiPSC lines. The TkDA3 human iPSC clone used in this study was provided by K. Eto and H. Nakauchi, University of Tokyo. Undifferentiated iPSCs were maintained in mTeSR1 medium on a dish coated with growth factor-reduced Matrigel. All cells were maintained at 37 °C in a humidified incubator with 5% CO2.

Hepatic lineage differentiation and LO differentiation

HLCs were differentiated from hiPSCs according to a published protocol [7], with minor modifications.

To generate hiPSC-LOs, hiPSC endoderm cells (250,000 cells), con-ECs (175,000 cells), and con-MCs (25,000 cells) or UC-derived ECs (UC-EC) (175,000 cells) and MCs (UC-MC) (25,000 cells) were cocultured in serum-free differentiation (SFD) medium containing epidermal growth factor (EGF, 10 ng/ml; Sigma-Aldrich), vascular endothelial growth factor (VEGF, 10 ng/ml; Life Technologies, Carlsbad, CA, USA), basic fibroblast growth factor (bFGF, 10 ng/ml; Wako Pure Chemical Industries), hepatocyte growth factor (HGF, 20 ng/ml; Sigma-Aldrich), and dexamethasone (100 nM; Sigma-Aldrich) in a three-dimensional (3D) microwell plate (Kuraray, Tokyo, Japan). The SFD medium contained 375 ml Iscove’s modified Dulbecco’s medium (Life Technologies), 125 ml Ham’s F-12 K medium (Life Technologies), 5 ml B27 supplement (Life Technologies), 2.5 ml N2 supplement (Life Technologies), 0.05% bovine serum albumin (Sigma-Aldrich), 2 mM l-glutamine (Life Technologies), 1% penicillin–streptomycin (Life Technologies), 0.45 mM monothioglycerol solution (Wako Pure Chemical Industries), and 0.5 mM l-ascorbic acid (Sigma-Aldrich). The hepatic lineage cells and LOs were differentiated and maintained at 37 °C in a humidified incubator with 5% CO2.

Macro-LO generation

Macro-LOs were generated from hiPSCs as described previously with minor modifications [19]. To generate macro-LOs, hiPSC endoderm (500,000 cells), con-ECs (350,000 cells), and con-MCs (50,000 cells) or UC-ECs (350,000 cells) and UC-MCs (50,000 cells) were resuspended in SFD medium containing EGF (10 ng/ml), VEGF (10 ng/ml), bFGF (10 ng/ml), HGF (20 ng/ml), and dexamethasone (100 nM) and were plated on presolidified growth factor-reduced Matrigel diluted with SFD medium (100 μl Matrigel + 100 μl SFD medium, incubated at 37 °C for at least 30 min to solidify) in a 48-well plate. Images of macro-LOs were taken at 0, 3, 12, 24, 48, and 72 h during formation. The macro-LO area and culture well area at each time point were quantified using ImageJ software (WS Rasband, ImageJ; NIH, Bethesda, MD, USA) and the following equation:

Percent area of LO = (LO area) / (Culture well area) × 100%.

The generated macro-LOs were cultured at 37 °C in a humidified incubator with 5% CO2.

Primary human hepatocyte culture

The dish-plated freshly isolated PHHs from humanized mice were purchased from PhoenixBio Co., Ltd (Higashihiroshima, Japan), without cryopreservation. The PHHs were cultured in hepatic growth medium (PhoenixBio). After 24 h of culture, PHHs were used for ALB and urea production analysis.

Transplantation of SDC-LOs into ALF mice

Alb-TRECK/SCID mice were a gift from the Tokyo Metropolitan Institute of Medical Science. The mice were bred and maintained according to the Yokohama City University institutional guidelines for the use of laboratory animals. All experimental procedures were approved by the institutional review board of the Animal Research Center, Yokohama City University School of Medicine (No. 075). The ALF model was generated as reported previously [20]. Briefly, 8–10-week-old mice were administered 1.5 μg/kg diphtheria toxin (DT) by intraperitoneal injection; 48 h later, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected using DRI-CHEM (Fujifilm, Tokyo, Japan) according to the manufacturer’s instructions. In vitro-generated and differentiated SDC-LOs (~1 × 106 hepatocytes) were collected and transplanted into the renal subcapsular space of each ALF mouse. The sham group received 50 μl of sterile saline.

RNA isolation, cDNA synthesis, and quantitative polymerase chain reaction

Total RNA was isolated using a PureLink viral RNA mini kit (Thermo Fisher Scientific, Waltham, MA, USA). Single-stranded cDNA was synthesized from RNA (1 μg) using a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific) according to the manufacturer’s instructions and was used for quantitative polymerase chain reaction (qPCR) with the specific primers and universal probe library probes presented in Additional file 1: Table S1. Target gene expression levels were calculated by the ΔΔCT method, with β-ACTIN serving as an internal control for normalization.

Flow cytometry

Cells were labeled with antibodies against cluster of differentiation (CD)31, CD146, CD144, CD90, CD45, CD73, CD105, HLA-DR, SSEA4, and TRA-1-60 (BD Biosciences) and analyzed by flow cytometry on a MoFlo Astrios system (Beckman Coulter, Fullerton, CA, USA).

ALB assay, cytochrome P450 3A4 assay, urea assay, and cell normalization

Human ALB levels were measured by enzyme-linked immunosorbent assay (ELISA) using a kit (Bethyl Laboratories, Montgomery, TX, USA). Urea production was evaluated using a QuantiChrom urea assay kit (BioAssay Systems, Hayward, CA, USA), and cytochrome P450 (CYP)3A4 activity was detected using a P450-Glo CYP3A4 assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

The total cell numbers in hiPSC-HLCs and hiPSC-LOs were normalized according to DNA amount. To calculate the cell number in LOs, we first counted the number of hiPSC-HLCs using an IN Cell Analyzer 2000 (GE Healthcare, Cardiff, UK) with Hoechst 33342 staining. Second, total DNA of hiPSC-LOs and hiPSC-HLCs was extracted using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany), and DNA was eluted in 50 μl of elusion buffer. Third, DNA concentration was determined using a nanodrop spectrophotometer (Thermo Fischer Scientific, Waltham, MA, USA). The total cell number in hiPSC-LOs was calculated using the following equation:

$$ mathrm{hiPSC}hbox{-} mathrm{LO}kern0.5em mathrm{cell}kern0.5em mathrm{number}kern0.5em =kern0.5em frac{left(mathrm{HLC}kern0.5em mathrm{cell}kern0.5em mathrm{number}right)kern0.5em times kern0.5em left(mathrm{LO}kern0.5em mathrm{DNA}kern0.5em mathrm{amount}right)}{left(mathrm{HLC}kern0.5em mathrm{DNA}kern0.5em mathrm{amount}right)} $$

ALB secretion, urea production, and CYP3A4 activity were also normalized to the calculated total cell number.

Periodic acid–Schiff staining and indocyanine green uptake and release

Glycogen was detected with the periodic acid–Schiff (PAS) staining kit (Muto Pure Chemicals, Tokyo, Japan) according to the manufacturer’s instructions. Cells were washed with PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. After washing with PBS, the cells were oxidized in 0.5% periodic acid solution for 7 min, then washed with PBS and incubated in Schiff reagent for 15 min. After three rounds of incubation for 2 min each in sulfurous acid water, the cells were washed with PBS and visualized by microscopy.

Dry indocyanine green (ICG) powder (Akorn, Buffalo Grove, IL, USA) (10 mg) was dissolved in 10 ml of hepatocyte culture medium (Lonza) to obtain a 1 mg/ml stock solution. The cells were cultured in ICG medium for 4 h at 37 °C, washed three times with PBS, and incubated in fresh medium for 4 h to determine ICG release.


SDC-LOs and kidney tissue transplanted with SDC-LOs were embedded in optimal cutting temperature compound (Sakura Finetek, Tokyo, Japan), and 7-μm sections were cut and mounted on MAS-GP type A-coated slides (Matsunami, Osaka, Japan). Tissue sections and cultured cells were fixed in a 4% paraformaldehyde solution in PBS for 10 min, washed three times with PBS, and blocked for 60 min with 10% enhanced chemiluminescence prime blocking agent in PBS containing 0.3% Triton X-100, followed by three washes with PBS. Samples were incubated overnight at 4 °C with antibodies against human ALB (Bethyl Laboratories), Hepatic nuclear factor (HNF)4A (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and α1-antitrypsin (A1AT) (Wako) in blocking buffer, washed three times with PBS, and incubated with a fluorophore-conjugated secondary antibody for 60 min at room temperature. The samples were washed three times in PBS and covered with mounting medium containing 4′,6-diamidino-2-phenylindole. Fluorescence was detected on an Axio Imager M1 microscope (Zeiss, Oberkochen, Germany).

Statistical analysis

Data are expressed as the mean ± standard error. The means of two groups were compared by the Mann–Whitney U test or unpaired t test. P < 0.05 was considered to indicate statistically significant differences. Data were obtained from at least three independent biological replicates, and analyses were performed with GraphPad Prism software (San Diego, CA, USA).