Animals
Male F344 rats (dipeptidyl-peptidase IV (DPPIV)+ strain; Sankyo Lab Service Corporation, Inc., Tokyo, Japan) and female F344 rats (DPPIV− strain; Charles River Japan, Yokohama, Japan) were used. All animals received proper care, and the Committee of Laboratory Animals approved the experimental protocol following guidelines stipulated by Sapporo Medical University (Approval No.: 17–032, 17–033, 17–034, 19–055, and 20–058). For GalN-induced injury in livers, GalN (Acros, Geel, Belgium, http://www.acros.com; 75 mg/100 g body weight dissolved in PBS) was intraperitoneally administered. For the transplantation experiment, female F344 rats (DPPIV+ strain; Charles River Japan, Yokohama, Japan, http://www.crj.co.jp) were administered two intraperitoneal injections of Ret (30 mg/kg body weight; Sigma-Aldrich, Co., St. Louis, MO, www.sigma-aldrich.com) two weeks apart [21, 27]. Two weeks after the second injection, 70% PH was performed. Sorted Thy1+ cells (DPPIV+ donor cells, 5 × 105 cells) and isolated EVs derived from Thy1+ cells were transplanted into Ret/PH livers (DPPIV− rat) through the spleen. Forty rats were randomly divided into control and 7 target groups (5 rats for each). Twenty-seven rats were used for the experiments of cell culture and transplantation. Therefore, the total number of rats used in this study was 67. The humane endpoints were established and monitored following guidelines stipulated by Sapporo Medical University. However, since any rats did not show the signs of the established endpoint in this study, all rats were euthanized by cutting inferior vena cava at PBS perfusion under anesthesia and the livers were resected for histological analyses. In all procedures of the treatments, rats were anesthetized using a mixture of O2/N2 (1:1) and isoflurane.
Sorting and culture of cells isolated from the liver
The detail of the isolation and subculture methods have been reported [20, 26, 28, 29]. Briefly, cells were isolated from DPPIV+ rats using the two-step collagenase-perfusion method [16]. After perfusion, the cell suspension was centrifuged at 50 × g for 1 min. The supernatant and precipitate were used for isolating Thy1+ cells and MHs, respectively. Thy1+ cells were isolated from injured livers 2 d after GalN-treatment (GalN-D3) [26]. SHs, MHs, SECs, and KCs were isolated from the liver of a healthy rat (8–12-week-old). The cells were cultured as described [20]. For cell sorting, mouse anti-rat Thy1 (Serotec, Raleigh, NC), mouse anti-rat SE-1 (Immuno-Biological Lab., Takasaki, Japan), and mouse anti-rat CD68 (Serotec) antibodies were used as primary antibodies (Supplemental Table 1). The cells were then sorted using magnetic cell sorting. Thy1+ cells were plated on 10-cm culture dishes. SECs and KCs were plated on a 24-well plate coated with rat-tail collagen. The number of viable cells was counted using the trypan blue exclusion test, and 1 × 105 cells/mL were plated on hyaluronic acid-coated dishes (Sigma-Aldrich Co., St. Louis, MO, 1 mg/35-mm dish). SHs were cultured for 10 d and then subcultured on Matrigel-coated 12-well plates as described [28, 29]. The cells were cultured in DMEM/F12 medium (Sigma-Aldrich) supplemented with 20 mM HEPES (Dojindo Chemical Laboratories, Kumamoto, Japan), 25 mM NaHCO3 (Kanto Chemical Co. Inc., Tokyo, Japan), 30 mg/L l-proline (Sigma-Aldrich), 0.1% bovine serum albumin (Serologicals Proteins Inc., Kankakee, IL), 10 mM nicotinamide (Sigma-Aldrich), 1 mM ascorbic acid 2-phosphate (Fujifilm Wako Pure Chem., Osaka, Japan), 10 ng/mL epidermal growth factor (EGF, BD Biosciences, Bedford, MA), ITS-X (BD Biosciences), 10− 7 M dexamethasone, and antibiotics. The medium was replaced every other day.
Separation of Thy1-MCs
Thy1+ cells derived from GalN-D3 were plated onto 10-cm culture dishes and cultured for 7 d. The proliferated cells consisted of epithelial cells and MCs (Fig. 1A). The epithelial cells form colonies resembling normal SHs and possess characteristics of LSPCs. These colonies were detached from the dish using the cell dissociation buffer at day 7 and collected. The MCs remaining on the dish were detached using 2% Trypsin/0.02% EDTA/PBS. Then, the number of viable cells was counted and epithelial cells and MCs (5 × 105 cells/mL) were transplanted to the Ret/PH model rat liver through the spleen. For isolating EVs, separated epithelial cells and MCs (1 × 106 cells) were seeded on 10-cm dishes and cultured in serum-free medium for 2 d. EVs were isolated from conditioned medium (CM).
Flow cytometry
Thy1+ cells were cultured for 7 d, collected by trypsinization, washed with PBS, and centrifuged at 150 × g for 5 min. The cells were then incubated with mouse anti-rat antibodies against CD90, CD73, and CD44 in DMEM containing 10% FBS for 30 min at 4°C; washed with PBS containing 2% FBS (wash buffer); centrifuged at 150 × g for 5 min; and incubated with rabbit anti-mouse IgG (H + L) antibodies conjugated with Alexa Fluor 488 in DMEM containing 10% FBS for 30 min at 4°C (Supplemental Table 1). Then, the cells were washed and centrifuged at 150 × g for 5 min. The pellet was suspended in a wash buffer containing propidium iodide solution and passed through a 35-µm cell strainer (Falcon, Corning Inc). The cells were analyzed on the FACSCanto flow cytometer (BD Biosciences, San Jose, USA). All antibodies used in this study are listed in Supplemental Table 1. The data were analyzed using the Kaluza Flow Cytometry Software version 1.1 (Beckman Coulter, Inc., Brea, USA).
Immunohistochemistry
The antibodies used in for immunohistochemistry are listed in Supplemental Table 1. Recipient rats were euthanized at 14 d after transplantation and their livers were immediately harvested and sliced on ice. Five-mm-thin sections were embedded in Tissue-Tek (Sakura Finetechnical Co., Tokyo, Japan), frozen in isopentane/liquid nitrogen, and stored at − 80°C until use. Some slices were fixed in 10% paraformaldehyde/buffered PBS. Enzyme- and immuno-histochemistry for DPPIV were performed to identify donor cells [16, 26]. SHPCs in Ret/PH-treated rat livers were identified as clusters comprising more than 10 small-sized hepatocytes, and the areas of SHPCs in the livers were measured using the cellSens Dimension software (OLYMPUS Corp., Tokyo, Japan).
Laser microdissection and gene expression analysis
Clusters of SHPCs in recipient livers were collected by using laser microdissection, following the manufacturer’s protocol [22, 30]. Total RNA was isolated from the captured cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Briefly, 7-µm-thin frozen sections were prepared from liver tissues and stained with hematoxylin. SHPC clusters were dissected under a microscope using an ultraviolet laser (MMI CellCut; Molecular Machines & Industries, Glattbrugg, Switzerland). The gene expression patterns were analyzed using quantitative real-time PCR (qRT-PCR).
miRNA extraction, miRNA microarray, and qRT-PCR
MicroRNAs (miRNAs) were extracted from EVs using the Qiazol lysis reagent and miRNeasy mini kit (Qiagen). A comprehensive analysis of miRNA expression was performed using the 3D-Gene miRNA Labeling kit and the 3D-Gene miRNA Oligo Chip (Toray Industries, Inc. Tokyo, Japan), which was designed to detect 727 miRNA sequences registered in miRBase release 20. For miRNA expression analysis, miRNAs were transcribed into cDNA using the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, USA) and RT primers provided with the TaqMan miRNA assay (Applied Biosystems). The cDNA products were analyzed using TaqMan miRNA sequence-specific probes for U6 small nuclear 2 (U6), miR-125b-5p, miR-145-5p, miR-199a-5p, miR-451-5p, miR-3473-5p, and let-7f, Premix Ex Taq (Takara), and the ABI Prism 7500 sequence detection system (Applied Biosystems) [30, 31]. The primers used are listed in Supplemental Table 2. All miRNA microarray data are registered in the GEO database (Accession No.
GSE222517).
Quantitative real-time PCR
For qRT-PCR, RNA was reverse-transcribed using the OmniScript RT Kit (Qiagen) and random hexamers as primers. qRT-PCR analyses were performed using TaqMan RNA sequence-specific probes and Premix Ex Taq (Takara). All qRT-PCR reactions were performed in triplicate in 96-well optical plates for all samples using the ABI Prism 7500 cycler (Applied Biosystems). The relative expression of each gene was normalized to the expression of Gapdh as a control. The primers used are listed in Supplemental Table 2.
Morphological analyses of cultured cells
The cultured cells were photographed using a phase-contrast microscope equipped with a CCD camera (Olympus Corp., Tokyo, Japan) to count the colonies and cells per colony. Ten fields per dish or well were selected randomly, at least three dishes or wells were examined per experiment, and at least two independent experiments were performed. All captured images were analyzed using the cellSens Dimension software (OLYMPUS Corp.).
Measurement of the labeling index
Cultured cells were treated with 40 µM 5-bromo-2ʹ-deoxyuridine (BrdU) for 18 h before fixation. The cells were fixed with absolute cold ethanol for 15 min and incubated first with 2 N HCl for 30 min at RT and then with 0.6% hydrogen peroxide in absolute methanol for 30 min at RT. The cells were blocked with BlockAce for 30 min at RT and incubated with a mouse anti-BrdU antibody for 60 min. The dishes were rinsed with PBS and subsequently incubated with a biotinylated anti-mouse antibody (Vector Laboratories, Burlingame, CA) for 30 min at RT. Next, the cells were incubated with an avidin–biotin complex solution (VECTASTAIN ABC kit; Vector Laboratories) and treated with 3,3-diaminobenzidine for color development. The number of cells with BrdU-positive nuclei was counted to determine the labeling index.
Isolation of EVs
EVs were separated from CM as reported [30]. Thy1+ cells were cultured for 5 d and washed with PBS and the medium was replaced with serum-free DMEM (Sigma-Aldrich Co.). After 48 h, CM was collected and centrifuged at 2,000 × g for 10 min at 4°C. The supernatant was filtered through a 0.22-µm filter (Millipore, Billerica, USA) to remove cellular debris. To prepare EVs, CM was ultracentrifuged at 110,000 × g for 70 min at 4°C [30, 31]. The supernatant was used as CM without EVs (CM-EVs) and the precipitate was re-suspended in 200 µL saline. The concentration of EVs was measured using the NanoDrop 1000 spectrometer (Thermo Fisher Scientific, Inc, Waltham, MA), and protein concentration was determined using a BCA assay kit (Thermo Fisher Scientific, Inc.).
Mass spectrometry
To evaluate the characterization of EVs, proteome analysis was performed by mass spectrometry. Aliquots containing 15 µg of total protein extracted from EVs were reductively alkylated with 10 mM dithiothreitol (Fujifilm Wako, Japan) followed by treatment with 20 mM iodoacetamide (Fujifilm Wako). The reductively alkylated proteins were digested with 0.75 µg of sequence grade trypsin/Lys-C (Promega, WI, USA) for 16 h at 37°C. The resultant peptides were desalted with a styrene divinylbenzene polymer tip column (GL Science, Tokyo, Japan). The desalted peptides were completely evaporated on a centrifugal evaporator. Finally, the obtained peptides were redissolved with 10 µL of ultrapure water containing 0.1% formic acid. This sample solution was subjected to Orbitrap Q Exactive Plus (Thermo Fisher Scientific, Inc.) through the EASY-nLC system equipped with an C18 column (0.075 mm × 125 mm, Nikkyo Technos, Tokyo, Japan). The sample was eluted with acetonitrile gradient from 0–30% in 90 min at a flow rate of 300 nL/min. All data were acquired in data-dependent mode and tandem MS (MS/MS) was performed by higher energy collision-induced dissociation. The MS/MS data were processed using the MaxQuant software 1.6.3.3 [32]. Peptide searches were performed with reference to the proteomic data of Rattus norvegicus obtained from UniProtKB (https://www.uniprot.org/). The mass spectrometry proteomics data are registered in the Proteome Xchange Consortium database (Accession No: PXD039384).
Transplantation of EVs into Ret/PH livers
Thy1+ cells were cultured for 7 d. Then, CM and the cells were used for isolating EVs and transplantation, respectively. The amount of EVs secreted by 5 × 105 donor Thy1+ cells in 48 h was quantified. The pellet obtained after ultracentrifugation of CM was resuspended in 200 µL saline and administered to the recipient liver through the spleen using a low dead-space syringe with a 21-gauge needle (NIPRO, Tokyo, Japan).
Transfection of cultured SHs with mimics
SHs cultured at 5 × 104 cells/well on Matrigel-coated 12-well plates were transfected with TaqMan miRNA mimics corresponding to miR-125b-5p (Applied Biosystems, MC), miR-145-5p (MC), miR-199a-5p (MC), miR-451-5p (MC), miR-3473-5p (MC), and miR-let-7f and the negative controls (Applied Biosystems, Cat No. 4464058) (Final concentration: 50 nM). The transfections were performed using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, USA) following the manufacturer’s instructions [30]. After 2 d, the medium was replaced, the cells were cultured for 5 d, and colony growth was evaluated.
Analysis of cytokine content
Isolated EVs were lysed in RIPA buffer containing 1 mM protease inhibitors (Sigma-Aldrich). The resulting lysates were assayed to detect Thy1-MCs-derived proteins using a custom-designed Quantibody rat-specific protein array (RayBiotech, Peachtree Corners, USA, cat. No. QAR-CAA-67) at Cosmo Bio, Ltd. (Tokyo, Japan) [30].
Overexpression of miR-199a-5p in EVs derived from Thy1+ cells using lentivirus
The transfections were performed using the XMIRXpress vector (SBI System Biosciences) according to the manufacturer’s instructions [26, 29]. Briefly, 293TN cells (3 × 106 cells) were plated on 75-cm2 culture flasks. Two µg of transfer plasmid (miR-125b-5p, miR-199a-5p, or non-target miRNA [NT]) and 20 µL pPACKH1-plasmid were mixed with 800 µL of serum-free DMEM in tubes and mixed by pipetting. Next, 24 µL of PureFection reagent (SBI System Biosciences) was added to tubes, vigorously vortexed, and incubated at room temperature for 15 min. The mixtures were added drop-wise into the flask and swirled to disperse evenly. After 2 d, media were collected into 12-mL tubes and centrifuged at 3,000 × g for 15 min to pellet cell debris. The viral supernatant was added to Thy1+ cell culture medium. EVs produced using transfected MCs were collected and evaluated for the expression of miR-125b-5p and miR-199a-5p by qRT-PCR. Their abilities to stimulate SHPC growth in Ret/PH model rats were examined as described.
Statistical analysis
Array data were analyzed using the MultiExperiment Viewer software. Microarray data were analyzed using Student’s t test. All other data were analyzed using Tukey’s multiple comparison test. Statistical analyses were performed using GraphPad Prism software (GraphPad Software, La Jolla, CA). Statistical significance was accepted at p < 0.05. The experimental results are expressed as mean ± standard error (SE).