Cell culture
Placentas at term (≥ 37 gestational weeks) were assembled from women without medical, obstetrical, and surgical complications. The collection of samples and their use for research purposes were approved by the Institutional Review Board (IRB) of CHA General Hospital, Seoul, Korea (IRB 07-18). All participating women provided written, informed consent. PD-MSCs were harvested as previously described [25]. PD-MSCs were maintained in alpha-modified minimal essential medium (α-MEM; HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA), 1% penicillin/streptomycin (P/S; Gibco), 25 ng/ml human fibroblast growth factor-4 (hFGF-4) (PeproTech, Rocky Hill, NJ, USA), and 1 µg/ml heparin (Sigma-Aldrich, St. Louis, MO, USA). Primary hepatocytes from seven-week-old male Sprague-Dawley (SD) rats (Orient Bio Inc., Seongnam, Korea) were isolated using a two-step collagenase perfusion process and were cultured in William’s E medium (Sigma-Aldrich) supplemented with 10% FBS (Gibco), 1% P/S (Gibco), and 4 mM L-glutamine (Gibco). To induce cholestatic injury in primary hepatocytes, 100 μM lithocholic acid (LCA) was applied for 12 h. Naïve and PD-MSCsPRL-1 (1x105 cells) were seeded onto Transwell inserts (8-µm pore size; Corning, NY, USA). Cells were maintained at 37 ℃ in a humidified atmosphere containing 5% CO2.
Gene transfections
The PRL-1 plasmid (human protein tyrosine phosphatase type 4 A, member 1; PTP4A1) was purchased (Origene Inc., Rockville, MD, USA) and used to induce the overexpression of the PRL-1 gene. The CMV6-AC vector containing PRL-1, the GFP reporter gene, CMV promoters and the antibiotic neomycin was digested with Sgf1 and Mlu1 restriction enzymes. The PRL-1 lentiviral plasmid was purchased from SeouLin Bioscience (Seongnam, Korea). The pLenti-RSV-EF1α vector including PRL-1 was constructed with a C-terminal GFP as well as the antibiotic puromycin. The AMAXA pCMV-GFP vector was obtained from Lonza (Basel, Switzerland). The resulting plasmid was confirmed by DNA sequencing. Naïve PD-MSCs (6x104 cells/cm2) were harvested and transfected by using an AMAXA system with a Human MSC Nucleofector Kit (Lonza) and lentiviral vector (SeouLin Bioscience). After transfections by each system, cells generated using AMAXA were selected by using 1.5 mg/ml neomycin. In addition, cells generated using lentiviral vector were selected by using 2 µg/ml puromycin for 7 days. We changed the medium every other day and observed changes in cell morphology. Cells were maintained at 37 ℃ in a humidified atmosphere containing 5% CO2.
Animal models and transplantation of MSCs
Seven-week-old male SD rats (Orient Bio Inc.) were housed under specific pathogen-free conditions. Liver cirrhosis was induced by common BDL as previously described [26]. Naïve cells (TTx naïve; n = 20) and PD-MSCsPRL-1 (TTx PRL-1+; n = 20) (2x106 cells, 9-10 passages) were stained with the PKH67 Fluorescent Cell Linker Kit (Sigma-Aldrich) and transplanted through the tail vein. Nontransplanted (NTx; n = 20) rats were maintained as well as sham controls (Con; n = 5). After 1, 2, 3 and 5 weeks, the rats were sacrificed, and their liver tissues and blood were harvested. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), and albumin levels were measured using separated serum from the blood (Southeast Medi-Chem Institute, Busan, Korea). In all animal experimental processes, protocols were approved by the Institutional Animal Care Use Committee (IACUC) of CHA University, Seongnam, Korea (IACUC-180023).
Histopathological and immunofluorescence analysis
To confirm the induction of liver cirrhosis with BDL and engraftment into target injured tissue for histological examination, liver specimens for each group (n = 5) were collected and fixed in 10% neutral buffered formalin (NBF). Samples were embedded in paraffin and OCT compound and processed as 5-μm thick sections for hematoxylin & eosin (H&E), Sirius red, Masson’s trichrome, and PKH67+ (green) staining. 4',6-Diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA, USA) was used as a counterstain for immunofluorescence. Morphometric images of whole sections in the liver were captured using a digital slide scanner (3DHISTECH Ltd., Budapest, Hungary).
Reverse transcription & quantitative real-time polymerase chain reaction (PCR) analysis
Total RNA was extracted from samples with TRIzol LS Reagent (Invitrogen). cDNA was synthesized by reverse transcription from total RNA (500 ng) using SuperScript III reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. To analyze stemness and hepatogenic differentiation markers of naïve and PD-MSCsPRL-1, PCR amplification was performed with specific primers (Table 1, designed by BIONEER, Daejeon, Korea). β-actin was used as an internal control.
To assess the differentiation, migration, mitochondrial biogenesis and albumin expression of samples, qRT-PCR was performed with human and rat primers (Tables 2 and 3, designed by BIONEER) and SYBR Green Master Mix (Roche, Basel, Switzerland) in a CFX Connect™ Real-Time System (Bio-Rad, Hercules, CA, USA). Target gene expression was normalized to GAPDH, and the 2-ΔΔCT method generated the relative values of mRNA expression. All reactions were performed in triplicate.
Western blotting
To assess the specific gene expression of PD-MSCsPRL-1 and cirrhotic liver tissues, samples were lysed in protein lysis buffer (Sigma-Aldrich). The protein lysates were loaded on sodium dodecyl sulfate polyacrylamide gels, and separated proteins were transferred to PVDF membranes. The following primary antibodies were used: anti-human PRL-1, anti-Oct4 (1:1,000; all from Abcam, Cambridge, UK), anti-albumin, anti-HLA-G (4h84) (1:1,000; all from Novus Biologicals, Littleton, CO, USA), anti-ATP5B (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), anti-ROCK1, anti-RhoA, anti-CDK4, anti-cyclin D1, and mitochondrial marker antibody sampler kit (1:1000; all from Cell Signaling Technology, Denvers, MA, USA). The loading control was anti-GAPDH (1:3,000; AbFrontier, Seoul, Korea). The following secondary antibodies were used: anti-mouse IgG (1:5,000; Bio-Rad, Hercules, CA, USA) and anti-rabbit IgG (1:10,000; Bio-Rad). The bands were detected using ECL reagent (Bio-Rad).
Multilineage differentiation analysis
To induce adipogenic and osteogenic differentiation, PD-MSCsPRL-1 (5 passages) generated using lentiviral and AMAXA systems were plated (5x103 cells/cm2) in each differentiation induction medium using a StemPro Adipogenesis and Osteogenesis differentiation kit (Gibco). Each medium was changed every other day. After approximately 21 days, cells were fixed with 4% paraformaldehyde and incubated for 1 h with Oil Red O (Sigma-Aldrich) for staining lipids and von Kossa with 5% silver nitrate (Sigma-Aldrich) to visualize the calcium deposits.
To induce hepatogenic differentiation, PD-MSCsPRL-1 (5 passages) were seeded (5x103 cells/cm2) in low-glucose Dulbecco’s modified Eagle medium (DMEM; HyClone) supplemented with 40% MCDB 201 medium (Sigma-Aldrich), 2% FBS, and 1% P/S coated with 5 µg/ml type IV collagen. After 48 h, the growth medium was replaced with low-glucose DMEM supplemented with 20 ng/ml epidermal growth factor (EGF; Peprotech), 10 ng/ml basic fibroblast growth factor (bFGF; Peprotech), 10 ng/ml bone morphogenetic protein-4 (Peprotech), 40% MCDB 201 medium, and 1% P/S. Then, PD-MSCsPRL-1 were cultured for an additional 48 h. The cells were treated for another week in step-1 medium consisting of low-glucose DMEM supplemented with 20 ng/ml hepatocyte growth factor (Peprotech), 10 ng/ml bFGF, 40% MCDB 201 medium, 2% FBS, and 1% P/S to progress to the maturation step. Then, differentiation was induced by incubating PD-MSCsPRL-1 for another week with step-2 medium consisting of low-glucose DMEM supplemented with 20 ng/ml oncostatin M (Peprotech), 1 µM dexamethasone (Sigma-Aldrich), 1× ITS+ Premix (Sigma-Aldrich), 40% MCDB 201 medium, 2% FBS, and 1% P/S. During steps 1 and 2, the culture media were changed every other day. After 18 days, each plate of PD-MSCsPRL-1 was incubated for 1 h at 37 ℃ with 1 mg/ml indocyanine green (ICG) (Dong in Dang Pharm., Siheung, Korea) uptake as described previously [25].
FACS analysis
PD-MSCsPRL-1 (5 passages) were stained with monoclonal antibodies specific for the following proteins to phenotype cell-surface antigens: CD34 (PE), CD90 (PE), HLA-ABC (FITC), HLA-DR (FITC) (BD Bioscience, San Diego, CA, USA), CD13 (PE) (BioLegend, San Diego, CA, USA), CD105 (PE) (R&D Systems, Minneapolis, MN, USA), and HLA-G (FITC) (Abcam). After staining, cells were washed in PBS and treated with appropriate isotype antibodies (BD Biosciences, San Jose, CA, USA). Cells were analyzed by a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). For each sample, at least 10,000 events were acquired.
Teratoma formation
To confirm teratoma formation by PD-MSCsPRL-1, nine-week-old male nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice (Laboratory Animal Research Center, Bungdang CHA Medical Center, Seongnam, Korea) were housed in an air-conditioned animal facility under pathogen-free conditions. A total of 5x105 cells of each cell type (lenti and AMAXA) were transplanted into one testis (Tx; n = 2). The other testis was not injected (Con; n = 2). After the testes were maintained for 14 weeks, the mice were sacrificed, and the testes of all groups were collected. Each section of testis tissue was stained with H&E.
Migration assay
Naïve and PD-MSCsPRL-1 (2x104 cells) were seeded into the upper inserts of a Transwell chamber (8-µm pore size; Corning) with or without siRNA-PRL-1 after 24 h to analyze the migration ability of naïve and PD-MSCsPRL-1. A scrambled siRNA (Invitrogen) was used as a negative control (NC). Each cell type was fixed with 100% methanol for 10 min and then stained with Mayer’s hematoxylin (Dako, Santa Clara, CA, USA) for 5 min. The number of stained cells was randomly counted in eight nonoverlapping fields on the membranes at a magnification of 200×. The experiments were performed in triplicate.
L-lactate production assay
To confirm the end product of glycolysis in naïve and PD-MSCsPRL-1, the lactate production rate was determined using a colorimetric L-lactate assay kit (Abcam). Cell lysates were deproteinized to eliminate endogenous LDH by a Deproteinizing Sample Preparation Kit–TCA (Abcam). Each sample was plated in a 96-well plate, and lactate reagent was added for 30 min. The absorbance was quantified using an Epoch microplate reader (BioTek, Winooski, VT, USA) at 450 nm. The lactate concentration was evaluated by the trend line equation. The experiments were conducted in triplicate.
Measurement of ATP production
To confirm that mitochondria provide energy, ATP concentrations of homogenized liver tissue samples, PD-MSCsPRL-1, and primary hepatocyte lysate were measured by an ATP determination kit (Thermo Fisher Scientific, Waltham, MA, USA). According to the manufacturer’s instructions, ATP concentrations were assessed using a microplate reader (BioTek) at 570 nm. The experiments were performed in triplicate.
Mitochondrial DNA (mtDNA) copy number assay
Genomic DNA (gDNA) was extracted from MSCs, primary hepatocytes, and homogenized BDL-injured rat liver to analyze mtDNA copy number. qRT-PCR amplification was conducted with specific primers (Table 2) containing 250 ng of gDNA, primers of nuclear DNA with FAM- and mtDNA with JOE-labeled quencher dye, and 1× TaqMan Universal Master Mix (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Data were obtained in triplicate.
XF Mito Stress assay
To further analyze mitochondrial metabolic functions in live PD-MSCsPRL-1, an XF24 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, USA) was used for real-time analysis of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Naïve and PD-MSCsPRL-1 were plated on XF24-well microplates (Seahorse Bioscience) (7x103 cells/well). The cells were adjusted for equilibrium in XF buffer for 60 min for contemporaneous analysis of OCR and ECAR by repeated cycles of mixing (3 min), incubation (2 min) and measurement (3 min) periods in a non-CO2 incubator. Following basal respiration measurements, the cells were sequentially treated with 0.5 µM oligomycin, 0.5 µM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and 1 µM rotenone/antimycin A (AA) mixture. The changes in respiration were recorded. The Seahorse XF24 Analyzer program was set according to the manufacturer’s recommendation. OCR/ECAR was then normalized by total cell number.
Statistical analysis
All data are expressed as the mean ± standard deviation (SD). Student’s t-test was used for analysis, and p values less than 0.05 were considered statistically significant. All experiments were conducted in triplicate.