Animals

F344 rats are inbred strains, and it is well known that immunological rejection of donor cells rarely occurs in cell transplantation experiments. Female F344/DuCrlCrlj rats (8 weeks old; Charles River Japan, Yokohama, Japan) were used as recipients. Male F344/Nslc rats (8–10 weeks old; Sankyo Lab Service Corporation, Inc., Tokyo, Japan) were used for obtaining donor cells. The F344/DuCrlCrlj strain genetically lacks dipeptidyl peptidase IV (DPPIV), whereas the F344/Nslc strain is wild and exhibits DPPIV expression. To identify donor cells in the recipient livers, we used DPPIV-positive cells isolated from F344/Nslc rat livers as donor cells and F344/DuCrlCrlj rats as the recipient. DPPIV-positivity can be demonstrated immunohistochemically and enzymohistochemically [28, 29]. All animals received appropriate care, and the experimental protocol was approved by the Committee of Laboratory Animals (Approval No.: 17-032, 17-033, 17-034, 19-055, and 20-058). The study was in line with the guidelines stipulated by Sapporo Medical University. For GalN-induced liver injury, GalN (75 mg/100 g body weight dissolved in phosphate-buffered saline [PBS]; Acros, Geel, Belgium, http://www.acros.com) was intraperitoneally administered. For the transplantation experiment, female F344 rats were administered two intraperitoneal injections of Ret (30 mg/kg body weight; Sigma-Aldrich, Co., St. Louis, MO, www.sigma-aldrich.com) at an interval of 2 weeks [22, 29]. Two weeks after the second injection, 70% PH was performed. Sorted Thy1+ (5 × 105) cells and isolated EVs obtained from cultured Thy1+ cells were administered to Ret/PH livers (DPPIV− rat) through the spleen. Forty rats were randomly divided into control and seven target groups (five rats per group). Twenty-seven rats were employed for cell isolation experiments. Accordingly, 67 rats were used in this study. The humane endpoints were established and monitored in accordance with the guidelines stipulated by Sapporo Medical University. No rats exhibited any signs of the established endpoints in this study. For histological analyses, rats were euthanized by incising the inferior vena cava via PBS perfusion under anesthesia, and the livers were resected for histological analyses. For all treatment methods, rats were anesthetized using a mixture of O2/N2 (1:1) and isoflurane.

Sorting and culture of cells isolated from the liver

The isolation and subculture methods have been described previously [21, 27, 28, 30, 31]. Briefly, liver cells were isolated from DPPIV+ rats using the two-step collagenase perfusion method [17], following which 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 3 days after GalN treatment (GalN-D3) [27]. SHs, MHs, SECs, and KCs were isolated from the liver of a healthy rat (8–12 weeks old). The cells were cultured as previously described [21] and then sorted via magnetic 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 for sorting Thy1+ cells, SECs, and KCs, respectively (Additional file 2: Table S1). SHs were isolated from a healthy liver as previously described [27]. After enumerating viable cells using the trypan blue exclusion test, cells were plated on each culture dish at a density of 1 × 105 cells/mL. Thy1+ cells were plated on 10-cm culture dishes coated with rat tail collagen. SECs and KCs were plated on a 24-well plate coated with rat tail collagen. Furthermore, SHs were plated on hyaluronic acid (Sigma-Aldrich Co., St. Louis, MO)-coated dishes (1 mg/35-mm dish). SHs were cultured for 10 days and then subcultured on Matrigel-coated 12-well plates as previously described [31, 32]. 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 (BD Biosciences, Bedford, MA), ITS-X (BD Biosciences), 10−7 M dexamethasone, and antibiotics. The medium was replenished every alternate day.

Separation of Thy1-MCs

The separation methods have been described previously [20, 21]. Thy1+ cells obtained after GalN-D3 treatment were seeded into 10-cm culture dishes and cultured for 7 days. The proliferated cells comprised epithelial cells and MCs (Fig. 1A). The epithelial cells formed colonies resembling normal SHs and exhibited the morphological characteristics of LSPCs. These colonies were detached and collected from the dish using a cell dissociation buffer on day 7. The remaining MCs on the dish were detached using 2% trypsin/0.02% EDTA/PBS. Subsequently, viable cells were enumerated, and epithelial cells and MCs (5 × 105 cells/mL) were transplanted into Ret/PH-treated rat livers through the spleen. For isolating EVs, separated epithelial cells and MCs (1 × 106 cells) were seeded into 10-cm dishes and cultured in a serum-free medium for 2 days. EVs were isolated from the conditioned medium (CM).

Fig. 1

Transplantation of Thy1-LSPCs and Thy1-MCs to Ret/PH-treated rat livers. A Schematic diagram showing the cell transplantation method. B Photos of SHPC clusters (black dotted lines) in hematoxylin–eosin (HE)-stained livers transplanted with control (left), Thy1-LSPCs (center), and Thy1-MCs (right). Inset depicts enlarged typical SHPCs (yellow square) in livers with and without Thy1+ cell transplantation. Fine fat droplets (empty vesicles) are observed in the cytoplasm of SHPCs. Scale bar, 500 μm. C The number of SHPC clusters per area and D area of the cluster were measured 14 days after transplantation. E Il17rb expression in SHPCs of livers that underwent Thy1+ cell transplantation was verified via qRT–PCR. Asterisks indicate significant differences at p < 0.05

Flow cytometry

To characterize Thy1+ cells, flow cytometry was performed as previously reported [32]. Briefly, Thy1+ cells were cultured for 7 days, trypsinized, and centrifuged at 150×g for 5 min. After removing the supernatant, the pellets were washed with PBS and incubated with mouse anti-rat antibodies against CD90, CD73, and CD44 in DMEM containing 10% FBS for 30 min at 4 °C. The pellets were collected and centrifuged at 150×g for 5 min. After washing with PBS containing 2% FBS (wash buffer), the pellets were 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 (Additional file 2: Table S1). The pellets were washed again after centrifugation. Subsequently, they were suspended in a wash buffer containing propidium iodide solution and were passed through a 35-µm cell strainer (Falcon, Corning Inc). The cells were analyzed using FACSCanto flow cytometer (BD Biosciences, San Jose, USA). All antibodies used in this study are listed in Additional file 2: Table S1. Data were analyzed using Kaluza Flow Cytometry Software version 1.1 (Beckman Coulter, Inc., Brea, USA).

Immunohistochemistry

The antibodies used for immunohistochemistry are listed in Additional file 2: Table S1. Recipient rats were euthanized 14 days 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 sections were fixed in 10% paraformaldehyde/buffered PBS. Enzyme- and immuno-histochemistry for DPPIV were performed to identify donor cells [20, 28]. SHPCs in Ret/PH-treated rat livers were identified as clusters containing > 10 small-sized hepatocytes, and areas covered by SHPCs in the livers were measured using cellSens Dimension software (OLYMPUS Corp., Tokyo, Japan).

Laser microdissection and gene expression analysis

SHPC clusters in recipient livers were obtained via laser microdissection, according to the manufacturer’s protocol [27, 32]. Total RNA was isolated from the collected cells using RNeasy Mini Kit (Qiagen, Hilden, Germany). Briefly, 7-μm-thick 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). Further, the gene expression patterns were analyzed using quantitative reverse transcription polymerase chain reaction (qRT–PCR).

MicroRNA (miRNA) extraction, microarray analysis, and qRT–PCR

Microarray analysis of miRNAs has been previously described [32]. miRNAs were extracted from EVs using Qiazol lysis reagent and miRNeasy mini kit (Qiagen). A comprehensive analysis of miRNA expression was performed using 3D-Gene miRNA Labeling kit and 3D-Gene miRNA Oligo Chip (Toray Industries, Inc. Tokyo, Japan), which was designed to detect 727 miRNA sequences registered in miRBase release 20. For gene expression analysis, miRNAs were transcribed into cDNA using TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, USA) and reverse transcription primers provided with TaqMan miRNA assay kit (Applied Biosystems). The cDNA products were analyzed using TaqMan miRNA sequence-specific probes for U6 small nuclear 2 (U6), miR-125b-5p (miR-125b), miR-145-5p (miR-145), miR-199a-5p (miR-199a), miR-451-5p (miR-451), miR-3473-5p (miR-3473), and let-7f Premix Ex Taq (Takara) and the ABI Prism 7500 sequence detection system (Applied Biosystems) [32]. The primers used in this study are listed in Additional file 2: Table S2. All miRNA microarray data are registered in the GEO database (Accession No. GSE222517).

qRT–PCR

The details of qRT–PCR analysis have been previously reported [27, 32]. Briefly, RNA was reverse-transcribed using 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). The reactions were performed in triplicate in 96-well optical plates for all samples using ABI Prism 7500 cycler (Applied Biosystems). The relative expression of each gene was normalized to that of the control GAPDH. The primers used in this study are listed in Additional file 2: Table S2.

Morphological analyses of cultured cells

The details of morphological analyses have been previously reported [27, 32]. The cultured cells were photographed under a phase-contrast microscope equipped with a CCD camera (Olympus Corp., Tokyo, Japan) to enumerate the colonies and cells per colony. After randomly selecting ten fields per dish or well, at least three dishes or wells were analyzed per experiment and at least two independent experiments were performed. All acquired images were analyzed using cellSens Dimension software (OLYMPUS Corp.).

Measurement of the labeling index (LI)

According to previous studies [27, 32], 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 room temperature (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 cells were washed with PBS and subsequently incubated with a biotinylated anti-mouse antibody (Vector Laboratories, Burlingame, CA) for 30 min at RT. Then, the cells were incubated with an avidin–biotin complex solution (VECTASTAIN ABC kit; Vector Laboratories) and treated with 3,3-diaminobenzidine for color development. Cells with BrdU-positive nuclei were enumerated to determine LI.

Isolation of EVs

EVs were separated from CM as previously described [32, 33]. Thy1+ cells were cultured for 5 days and washed with PBS, and CM was replaced with serum-free DMEM (Sigma-Aldrich Co.). After 48 h, CM was collected and centrifuged for 10 min at 2000×g and 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 for 70 min at 110,000×g and 4 °C [32, 33]. The supernatant contained CM without EVs (CM-EVs), and the precipitate was resuspended in 200 µL of saline. The concentration of EVs was measured using NanoDrop 1000 spectrometer (Thermo Fisher Scientific, Inc, Waltham, MA), and protein concentrations were determined using BCA assay kit (Thermo Fisher Scientific, Inc.).

Mass spectrometry (MS)

To characterize EVs, proteome analysis was performed using MS. 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 using a centrifugal evaporator. Finally, the obtained peptides were redissolved in 10 μL of ultrapure water containing 0.1% formic acid. This sample solution was subjected to MS using Orbitrap Q Exactive Plus (Thermo Fisher Scientific, Inc.) coupled to the EASY-nLC system equipped with a C18 column (0.075 mm × 125 mm, Nikkyo Technos, Tokyo, Japan). The sample was eluted using an acetonitrile gradient from 0 to 30% within 90 min at a flow rate of 300 nL/min. All data were obtained in a data-dependent mode, and tandem MS (MS/MS) was performed based on high energy collision-induced dissociation. The MS/MS data were processed using MaxQuant software 1.6.3.3 [34]. Peptide searches were performed by referring to the proteomic data of Rattus norvegicus obtained from UniProtKB (https://www.uniprot.org/). The proteomic data are registered in the Proteome Xchange Consortium database (Accession No: PXD039384).

Administration of EVs to Ret/PH livers

Thy1+ cells were cultured for 7 days. Then, the cells and CM were used for transplantation and EV isolation, respectively. The number of EVs secreted by 5 × 105 donor Thy1+ cells in 48 h was estimated. The pellet obtained after ultracentrifugation of CM was resuspended in 200 µL of saline and administered to recipient livers through the spleen using a low dead-space syringe with a 21-gauge needle (NIPRO, Tokyo, Japan) [32].

Transfection of miRNA mimics into cultured SHs

SHs cultured on Matrigel-coated 12-well plates at a density of at 5 × 104 cells/well were transfected with TaqMan miRNA mimics corresponding to miR-125b-5p (Applied Biosystems, MC), miR-145-5p (miR-145), miR-199a-5p (miR-199a), miR-451-5p (miR-451), miR-3473-5p (miR-3473), and miR-let-7f as well as negative controls (final concentration: 50 nM; Applied Biosystems, Cat No. 4464058). Transfection was performed using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions [32]. After 2 days, the medium was replenished, cells were cultured for 5 days, 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-MC-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) [32].

Overexpression of miR-199a-5p in EVs derived from Thy1+ cells using lentivirus

Transfection was performed using XMIRXpress vector (SBI System Biosciences) according to the manufacturer’s instructions [32, 35]. Briefly, 293 TN (3 × 106) cells were inoculated into 75-cm2 culture flasks. Then, 2 µg of transfer plasmid (miR-125b-5p, miR-199a-5p, or nontarget miRNA) and 20 µL of pPACKH1 plasmid were mixed with 800 µL of serum-free DMEM in tubes via pipetting. Next, 24 µL of PureFection reagent (SBI System Biosciences) was added to the tubes, vortexed vigorously, and incubated at RT for 15 min. The mixtures were added dropwise into the flask and swirled to disperse evenly. After 2 days, media were collected in 12-mL tubes and centrifuged at 3000×g for 15 min to remove cell debris. The viral supernatant was added to the culture medium. EVs produced via transfected MCs were collected and analyzed for the expression of miR-125b-5p and miR-199a-5p using qRT–PCR. The ability of EVs to induce SHPC growth in Ret/PH rat models was analyzed.

Effects of miR-199a mimics as well as CM derived from SECs treated with CINC-2 and KCs treated with Thy1-EVs on SH growth

SECs and KCs isolated from a healthy rat liver were cultured on type I collagen-coated 12-well plates (Corning Inc). Three hours after seeding, the culture medium of SECs and KCs was replaced with a medium containing CINC-2 or Thy1-EVs and miR-199a mimics, respectively. These cells were cultured for 2 days, and CM was obtained. Next, 100 μL of CM was added to the SH culture medium, and SHs were then cultured for 7 days. CINC-2-treated SEC-CM was continuously added for 7 days. Contrarily, KC-CM treated with Thy1-EVs was added only for the initial 2 days. The size of SH colonies was measured 7 days after plating.

Statistical analysis

Array data were analyzed using MultiExperiment Viewer software (https://mev.tm4.org/). Microarray data were analyzed using Student’s t test. All other data were compared using Tukey’s multiple comparison test. Statistical analyses were performed using GraphPad Prism software (GraphPad Software, La Jolla, CA). A p value of < 0.05 was considered to indicate statistical significance. The experimental results are expressed as mean ± standard error.