Six-week-old Sprague-Dawley rats were obtained (Orient Bio Inc.) and maintained in an air-conditioned facility. Hepatic failure was induced CCl₄ (approximately 1.6 g/kg; Sigma-Aldrich) dissolved at 0.8 mg/ml in corn oil and intraperitoneally injected at 0.2 ml/100 g body weight twice a week for 3 weeks. Control rats (n=5) were injected with an equal volume of corn oil alone. PD-MSCs (2×10⁶ cells, 8∼10 passages) were injected into the tail vein of rats (TTX; n=19). Non-transplanted (NTX; n=19) rats were maintained as sham controls. Liver tissues were harvested at 1, 2 and 3 weeks from rats in all groups. All animal experimental processes and the experimental protocol were approved by the Institutional Animal Care and Use Committee of CHA University, Seongnam, Korea (IACUC-140009).

Equal amounts of serum from individual animals were pooled (n=5). To isolate exosomes from rat serum, we used ExoQuick Exosome Precipitation solution (System Biosciences). The solution for exosome precipitation was used according to the manufacturer’s instructions.

All participants provided written and informed consent prior to sample collection. Placental tissues were collected from women who had no medical, obstetrical, and surgical complications and who delivered at term (37 gestational weeks). The collection of samples and their use for research purposes were approved by the IRB of CHA General Hospital, Seoul, Korea (IRB 07-18). PD-MSCs were harvested as previously described. PD-MSCs were collected from the inner side of the chorionic membrane of the placenta. The cells scraped from the membrane were treated with 0.5% collagenase IV (Sigma-Aldrich) and cultured in Ham’s F-12/Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 1% penicillin/streptomycin (Pen-Strep, Gibco), 25 ng/ml human fibroblast growth factor-4 (Peprotech), and 100 μg/ml heparin (Sigma-Aldrich). Rat epithelial cells (WB-F344s) were cultured in α-MEM (Gibco) supplemented with 1% Pen-Strep and 5% FBS. Human umbilical vein endothelial cells (HUVECs) were cultured in endothelial cell growth medium (EGM)-2 SingleQuots (Lonza) at 37℃ in a 5% CO₂ incubator.

WB-F344s (2×10⁵) and HUVECs (6×10⁴) were seeded onto cover glasses (Marlenfeld GmbH & Co.), and for double cell co-culture experiments, WB-F344s (2×10⁵) were seeded onto cover glasses in a 100 mm culture plate (Corning). After 3 hours, the cells were exposed to 3 mM CCl₄ for 24 hours. The next day, the cells on cover glass were transferred to 6-well plates and co-cultured with PD-MSCs (6×10⁴) were in Transwell inserts (3 μm pore).

To sort co-cultured WB-F344s and HUVECs, we performed MACS analysis. Harvested WB-F344s and HUVECs were incubated with biotin-conjugated anti-CD31 (Miltenyi Biotec Inc.). Anti-biotin microbeads (Miltenyi Biotec Inc.) were reacted with the collected cells. The bound cells were sorted using MidiMACS Starting Kits (Miltenyi Biotec Inc.).

Total RNA was isolated from rat liver tissues and cells using TRIzol (Invitrogen). Reverse transcription was performed with 500 ng total RNA and Superscript III reverse transcriptase (Invitrogen). Real-time PCR was performed using SYBR EX Taq (Roche) and an Exicycler^TM 96 quantitative thermal block (Bioneer). PCR conditions were as follows: denaturation at 95℃ for 15 minutes and 20 seconds; 40 cycles at 95℃ for 30 seconds; annealing at 52∼60℃ for 40 seconds; extension at 70℃ for 15 minutes; and a final extension at 72℃ for 7 minutes. Gene expression was normalized to the expression of GAPDH. The sequences of the primers are as follows: rat CRP forward 5’- GCT TTT GGT CAT GAA GAC ATG TC -3’, rat CRP reverse 5’- TCA CAT CAG CGT GGG CAT AG -3’, rat Axin2 forward 5’- AAA CCT ATG CCT GTC TCC TC -3’, rat Axin2 forward 5’- ATC CAC ACA TTT CTC CCT CT -3’, rat VEGF forward 5’- ACG GAC AGA CAG ACA GAC AC -3’, rat VEGF reverse 5’- CTT CTG GGC TCT TCT TCT CTC TC -3’, rat albumin forward 5’- GCC CCA GAA CTC CTT TAC TA -3’, rat albumin reverse 5’- AAT CTC TGC ATA CTG GAG CA -3’, rat GAPDH forward 5’- TCC CTC AAG ATT GTC AGC AA -3’, rat GAPDH reverse 5’- AGA TCC ACA ACG GAT ACA TT -3’, human CRP forward 5’- TCG TAT GCC ACC AAG AGA CAA GAC A -3’, human CRP reverse 5’- AAC ACT TCG CCT TGC ACT TCA TAC T -3’, human Axin2 forward 5’- TCC CCA CCT TGA ATG AAG AA -3’, human Axin2 reverse 5’- TGG TGG CTG GTG CAA AGA -3’, human VEGF forward 5’- GCC TTG CCT TGC TGC TCT AC -3’, human VEGF reverse 5’- ACA TCC ATG AAC TTC ACC ACT TCG -3’, human albumin forward 5’- TGA GAA AAC GCC AGT AAG TGA C -3’, human albumin reverse 5’- TGC GAA ATC ATC CAT AAC AGC -3’, human GAPDH forward 5’- GCA CCG TCA AGG CTG AGA AC -3’, and human GAPDH reverse 5’- GTG GTG AAG ACG CCA GTG GA -3’. All reactions were conducted in duplicate or triplicate. Relative mRNA expression was analyzed by the comparative CT method.

Rat liver tissues and cells were lysed in RIPA buffer (Sigma-Aldrich) supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitor (Sigma-Aldrich). A total of 45 μg protein was separated in sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE). The separated proteins were transferred onto PVDF membranes (Bio-Rad). The membranes were incubated with anti-CRP (1：1,000, Abcam Inc.), anti-VEGF (R&D system), anti-VEGFR2 (R&D system), anti-β-catenin (active form, Cell Signaling), anti-pGSK3 (Cell Signaling), anti-β-catenin (active form, Cell Signaling), anti-albumin (Novus), anti-HNF1α (Abcam Inc.), anti-cyclinD1 (AbFrontier), anti-actin (Sigma-Aldrich), anti-tubulin (Abcam Inc.), and anti-GAPDH (AbFrontier) antibodies at 4℃ over-night. The membranes were then incubated with horseradish peroxidase-conjugated secondary anti-mouse IgG (Cell Signaling), anti-rabbit IgG (Cell Signaling) or anti-goat IgG (Santa Cruz) antibody for 1 hour at RT. Bands were detected using Clarity Western ECL kit (Bio-Rad).

To observe the degree of inflammation in tissues following transplantation with PD-MSCs or not (NTX), we used anti-NF-κB antibody. The sections were incubated in 3% H₂O₂ in methanol to block endogenous peroxidase activity. After antigen retrieval, the slides were incubated with an anti-NF-κB antibody (Santa Cruz) at 4℃ over-night, followed by an hour incubation with biotinylated secondary anti-rabbit antibody at room temperature (RT). Incubation with horseradish peroxidase-conjugated streptavidin–biotin complex (Dako) and 3,3-diaminobenzidine (EnVision Systems) was performed to generate a chromatic signal. The samples were counterstained with Mayer’s hematoxylin. Additionally, the percentage of hepatocytes with NF-κB-positive nuclei relative to the total number of hepato-cytes was counted in randomly selected sections (three fields per rat at ×400 magnification). Images were detected using a Zeiss Axioskop 2 MAT microscope (Carl Zeiss MicroImaging).

To analyze the localization of VEGF and active β-ca-tenin in liver tissues, we embedded rat livers in Tissue-Tek OCT compound (Sakura Finetechnical Co. Ltd.) and stored at −80℃. Cryosections of tissues were fixed in methanol (MERCK) and incubated in blocking solution (Dako). Anti-VEGF (Novus) and anti-active β-catenin (Cell Sig-naling) antibodies were used as primary antibodies at 4℃ over-night. The secondary antibodies, Alexa 488 and 569 (Invitrogen), were incubated.

To analyze the localization of β-catenin and BrdU in rat hepatocytes, the WB-F344s co-cultured with PD-MSCs were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton X-100 and 3% H₂O₂. The cells were incubated with blocking solution (Dako) at RT and rabbit anti-β-catenin (Cell Signaling) and mouse anti-BrdU (Cell Signaling) antibodies at 4℃ over-night. Subsequently, the cells were incubated with Alexa 568- or 488-conjugated secondary antibodies (Invitrogen) at RT. The slides were counterstained with DAPI. The images were observed with a fluorescence microscope EVOS (Thermo Fisher Scientific). All experiments were performed in triplicate.

The concentrations of interleukin (IL)-6, IL-10 and CRP were determined by enzyme-linked immunosorbent assay (ELISA). Equal amounts of serum from individual animals or culture media were pooled (n=5). All reactions were performed in triplicate. IL-6 and IL-10 concentrations were assayed using rat IL-6 and IL-10 ELISA kits (R&D system), according to the manufacturer’s protocols. The concentration of CRP was measured using a rat C-Reactive Protein ELISA kit (Invitrogen) according to the manufacturer’s instructions. The experiments were performed in triplicate.

Statistical significance was analyzed using Student’s t-test with a significance level of p<0.05, and data were represented as the mean±standard deviation (SD). For multivariate data analysis, group differences were assessed with two-way ANOVA, followed by Tukey HSD test. All experiments were conducted in duplicate or triplicate.

To evaluate the anti-inflammatory effects of PD-MSC transplantation, we analyzed NF-κB expression in CCl₄-injured rat livers using immunohistochemistry. As shown in Fig. 1A, in the CCl₄-damaged NTX group, NF-κB was excessively localized in the nuclei for up to 3 weeks; however, transplantation of PD-MSCs resulted in a marked reduction in NF-κB localization. Moreover, the number of NF-κB-positive cell was significantly lower in the TTX group than in the NTX group (*, p<0.05, Fig. 1B). Furthermore, we assayed the concentrations of IL-10, an anti-inflammatory factor, and IL-6, a regeneration factor, in the rat serum via ELISA. Compared to that in the NTX group, the expression of IL-10 in the rat serum was significantly increased in the TTX group (*, p<0.05, Fig. 1C). The secreted form of IL-6, which functions in hepatic regeneration, was also significantly increased in the TTX group (*, p<0.05, Fig. 1D).

Similar to IL-10 and IL-6, the expression of CRP in serum (Fig. 2A) and total liver protein (Fig. 2C) progressively increased in the TTX group compared to that in the control and NTX groups of in the CCl₄-injured rat model. Additionally, CRP in exosomes isolated from serum was upregulated in the TTX group when normalized to heat shock protein (HSP)-70 and HSP-90, which are internal markers of exosomes (Fig. 2B and 2D). Therefore, PD-MSCs attenuate inflammatory responses and promote anti-inflammation through the induction of CRP expre-ssion.

To analyze the correlation between Wnt signaling and angiogenesis, we confirmed the expression and localiza-tion of the factors related to angiogenesis and Wnt sig-naling. Compared to those in the control and NTX groups, the factors related to angiogenesis, such as VEGF and VEGFR2, were significantly induced in the TTX group (*, p<0.05, Fig. 3A, Supplementary Fig. S1A, and S1C). However, the expression of pGSK3, a Wnt inhibitory molecule, was significantly decreased in the TTX group compared to that in the control and NTX groups (*, p<0.05, Fig. 3B, Supplementary Fig. S1B). In contrast to GSK3, the active form of β-catenin was upregulated by PD-MSCs in CCl₄-injured rat livers (Fig. 3B, Supplementary Fig. S1D). To investigate the localization and expression of VEGF and β-catenin, we performed immunofluorescence. VEGF and active β-catenin were localized in the cytoplasm and nuclei, respectively. Additionally, the expre-ssion of VEGF and β-catenin was higher in the control and TTX groups than in the NTX group (Fig. 3C). These results mean that activated β-catenin by PD-MSCs transplantation translocates into nucleus following promotes expression of VEGF in CCl₄-injured rat livers. Further, to demonstrate the interaction between CRP, Wnt signaling, and angiogenesis in the liver, we analyzed the correlation between CRP, β-catenin, and VEGF of normalized fold change by band intensity of western blot. The correlation coefficient (R²) of CRP and β-catenin was 0.8992 (Fig. 3D) as well as that of β-catenin and VEGF was 0.9295 (Fig. 3E). Therefore, these results represent that CRP, Wnt pathway, and vascular remodeling strongly correlate with one another in CCl₄-injured rat liver.

To demonstrate the correlation among CRP, Wnt, and angiogenic and hepatic regeneration factors in hepato-cytes, we transfected siRNA-CRP in rat hepatocytes and analyzed the mRNA and protein levels of CRP, albumin and factors related to Wnt signaling and angiogenesis. The expression of CRP, Axin2 (Fig. 4B), VEGF, and albumin (ALB) (Supplementary Fig. S2) mRNA was lower in siRNA-CRP-transfected hepatocytes than in non-transfected hepatocytes. As assessed by western blotting, CRP and VEGF protein expression was significantly suppressed via siRNA-CRP transfection (*, p<0.05, Fig. 4A and 4B). Additionally, the expression of β-catenin, a key factor in the Wnt pathway, was significantly inhibited by siRNA-CRP transfection in rat hepatocytes (*, p<0.05, Fig. 4C). Also, hepatic regeneration and proliferation factors such as ALB, hepatic nuclear factor 1 alpha (HNF1α), and cyclinD1 were significantly repressed by siRNA-CRP transfection in WB-F344s (*, p<0.05, Fig. 4D∼F). Therefore, CRP directly regulates angiogenesis through the Wnt pathway and promotes hepatic regeneration.

To demonstrate the effect of CRP induced by PD-MSCs, we cultured CCl₄-treated rat hepatocytes with PD-MSCs. The expression of CRP at both mRNA and protein levels was upregulated in rat hepatocytes by co-culture with PD-MSCs (Fig. 5A and 5B). To verify cell proliferation and localization of β-catenin, we performed immunofluorescence staining for BrdU and active β-ca-tenin in rat hepatocytes that were co-cultured with PD-MSCs post-CCl₄ treatment. The localization of BrdU and β-catenin in rat hepatocytes is shown in Fig. 5C. The number of BrdU- or β-catenin-positive cells was significantly higher in the CCl₄-treated and PD-MSC co-cultured group than in the non-co-cultured group (*, p<0.05, Fig. 5D). Also, the protein level of HNF1α and cyclinD1, hepatic regeneration and proliferation markers, was dramatically decreased in CCl₄ treated WB-F344s while their expression were recovered by PD-MSCs co-cultivation (*, p<0.05, Fig. 5E∼G). Therefore, CRP upregulation by PD-MSCs in rat hepatocytes activates Wnt signaling and facilitates hepatocyte regeneration.

To demonstrate the interaction between hepatocytes and ECs via CRP and the Wnt signaling pathway, we used the culture scheme shown in Fig. 6A. The expression of CRP, VEGF, Axin2 and albumin was analyzed in hepatocytes, WB-F344s, and ECs, HUVECs. In sorted hepatocytes and ECs, CRP mRNA was upregulated by co-culture with PD-MSCs (Fig. 6B and 6C). Additionally, the expression of VEGF and Axin2 was increased in CCl₄-treated and PD-MSC co-cultured hepatocytes and ECs (Fig. 6D and 6E, Supplementary Fig. S3A and S3B). Furthermore, albumin, a hepatocyte marker, was upregulated in hepatocytes co-cultured with PD-MSCs (Supplementary Fig. S3C). However, albumin was not detected in the sorted ECs (Supplementary Fig. S3D). The expression of CRP protein, as detected by western blotting, was increased in the CCl₄-treated and PD-MSC co-cultured rat hepatocytes (Supplementary Fig. S4A and S4B). Furthermore, the Wnt pathway (Supplementary Fig. S4A, S4C and S4E), angiogenesis (Supplementary Fig. S4A and S4F) and hepatic regeneration (Supplementary Fig. S4A and S4D) were activated in the co-cultured group. Comparing between only hepatocytes culture and EC cells and hepatocytes on co-culture systems, there was no difference between liver regeneration by expression of albumin. However, the difference between vascular regeneration and Wnt signaling factors was more dynamic on co-culture systems of EC cells and hepatocytes because the VEGF secretion is exceptionally shown in the EC rather than the WB-F344. Therefore, we finally co-cultivated EC and hepatocytes to study hepatic regeneration by vascular regeneration and demonstrated CRP increased by PD-MSCs co-cultivation prompts vascular regeneration through Wnt signaling in hepatocytes by interacting with ECs.