PIN1 Suppresses the Hepatic Differentiation of Pulp Stem Cells via Wnt3a

Abstract

This study aimed to investigate the role of PIN1 on the hepatic differentiation of human dental pulp stem cells (hDPSCs) and its signaling pathway, as well as the potential therapeutic effects of hDPSC transplantation and PIN1 inhibition on CCl₄ (carbon tetrachloride)–induced liver fibrosis in mice. The in vitro results showed that hepatic differentiation was suppressed by infection with adenovirus-PIN1 and promoted by PIN1 inhibitor juglone via the downregulation of Wnt3a and β-catenin. Compared with treatment with either hDPSC transplantation or juglone alone, the combination of hDPSCs and juglone into CCl₄-injured mice significantly suppressed liver fibrosis and restored serum levels of alanine transaminase, aspartate transaminase, and ammonia. Collectively, the present study shows for the first time that PIN1 inhibition promotes hepatic differentiation of hDPSCs through the Wnt/β-catenin pathway. Furthermore, juglone in combination with hDPSC transplantation effectively treats liver fibrosis, suggesting that hDPSC transplantation with PIN1 inhibition may be a novel therapeutic candidate for the treatment of liver injury.

Keywords

NIMA-interacting peptidylprolyl isomerase dental pulp differentiation hepatocytes Wnt3a liver fibrosis

Introduction

Currently, cell-based therapies are emerging as an alternative to whole organ transplantation (Schuppan and Afdhal 2008; Wallace et al. 2008). Stem cell therapies based on hematopoietic stem cells, bone marrow–derived mesenchymal stem cells, and adipose tissue–derived mesenchymal stem cells have been successfully used in preclinical animal studies and clinical studies on patients with acute liver failure or chronic liver disease with end-stage cirrhosis (Kisseleva et al. 2010; Cho et al. 2011). However, methods used to harvest stem cells for these purposes are highly invasive and painful for the donor. Furthermore, the number and differentiation potential of stem cells decline with increasing age (Martens et al. 2013). Therefore, one alternative transplantable source of hepatic progenitors for the treatment of liver disease is subject to intensive investigation.

Human dental pulp contains human dental pulp stem cells (hDPSCs), which can differentiate into cells of odontogenic, adipogenic, osteogenic, chondrogenic, myogenic, and neurogenic lineages in vitro (Iohara et al. 2006). One advantage of hDPSCs is that they can be easily obtained noninvasively from teeth without any further surgical intervention with respect to the donor. The successful transplantation or engraftment of hDPSCs into adult animals, such as mice, rats, and rabbits, has been reported (de Mendonca et al. 2008; Huang et al. 2008; Gomes et al. 2010). Importantly, hDPSCs can differentiate into hepatocyte-like cells, which exhibit hepatocytic morphology (Ishkitiev et al. 2010; Ishkitiev, Calenic, et al. 2012; Ishkitiev, Yaegaki, et al. 2012). Taken together, these reports suggest that hDPSCs are an alternative source for cell therapy–based treatments of liver cirrhosis.

PIN1, or protein interacting with NIMA (never in mitosis A)–1, is a peptidyl-prolyl isomerase that can alter the conformation of phosphoproteins, thereby affecting protein function (Lu et al. 2007). PIN1 regulates a variety of cellular events for cell cycle progression, differentiation, and stress responses (Ryo et al. 2003). We recently reported that PIN1 inhibition can promote the odontogenic differentiation of hDPSCs (Lee et al. 2014) but suppress osteoclast differentiation (Cho et al. 2015).

There are many kinds of liver diseases, including hepatitis (viral infection–mediated inflammation of liver), fascioliasis (parasitic infection–mediated hepatic disease), and steatohepatitis (fatty liver disease, alcoholic/nonalcoholic). Although liver transplantation has long been regarded as an effective treatment strategy, the growing knowledge of cell biology has exploited an alternative stem cell based–cell therapy for patients with liver diseases (Vosough et al. 2011; Kadyk et al. 2015). One of the most common liver diseases, nonalcoholic steatohepatitis (NASH), is managed solely by lifestyle changes (e.g., diet and exercise) because it currently has no obvious treatment methods in spite of the increasing prevalence rate of NASH in the developed countries (Nakatsu et al. 2012).

PIN1^–/– mice are highly resistant to NASH, suggesting that PIN1 inhibition could provide a novel therapeutic strategy for it (Nakatsu et al. 2012). In contrast, PIN1^–/– mice exhibit more liver necrosis after ischemia/reperfusion injury when compared with wild-type mice (Kuboki et al. 2009), and PIN1 overexpression attenuates acute liver injury (liver necrosis) induced by carbon tetrachloride (CCl₄) in mice (Risal et al. 2012). Although results from these studies are inconsistent depending on types of liver injury, PIN1 seems to play a critical role in liver disease and/or regeneration.

To provide a novel strategy for liver fibrosis, this study aims to investigate the role of PIN1 in the hepatic differentiation of hDPSCs with its underlying signaling pathways and to determine whether hepatocyte-like cells derived from hDPSCs and PIN1 inhibition can be useful for liver repair or regeneration in the CCl₄-injured liver fibrosis of mice.

Materials and Methods

Cell Culture of hDPSCs

Human dental pulp tissues were obtained from clinically healthy permanent teeth (premolars) extracted during orthodontic treatment. All the human sample experiments were approved by the institutional review board and the research ethics committee of Kyung Hee University. hDPSCs were isolated and cultured as previously described (Ishkitiev et al. 2010; Lee et al. 2014). In brief, extracted primary cells cultured within the first 5 passages were expanded in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum with 1% antibiotic and subsequently submitted to magnetic-activated cell sorting to isolate hDPSCs. CD34⁺/c-kit⁺/STRO-1⁺ cells were defined as hDPSCs. To induce hepatic differentiation, primary hDPSCs were cultured in hepatogenic medium (HM) containing DMEM, 2% fetal calf serum, and 20 ng/mL of rh-hepatocyte growth factor for 5 d, followed by a mixture of 10 ng/mL of Oncostatin M, 10 nmol/L of dexamethasone, and 1% ITS-X for an additional 15 d, as described previously (Ishkitiev et al. 2010). Hepatic differentiation of hDPSCs was induced in the presence or absence of juglone or vehicle (ethanol, <1% final concentration).

Preparation of Recombinant PIN1 Adenovirus

Adenoviruses encoding PIN1 (Ad-PIN1) or β-galactosidase that were constructed with the ViraPower Adenovirus Expression System (Invitrogen) were kindly provided by Professor Byung-Hyun Park (Jeonbuk National University). Adenoviral infection was carried out at a multiplicity of infection of 100 in OPTI-MEM (Gibco BRL Co.) for 5 h before commencement of hepatogenic induction.

Periodic Acid–Schiff Staining

Cells were fixed in 4% paraformaldehyde for 3 min and then oxidized in 1% periodic acid for 5 min, washed with phosphate-buffered saline, and treated with Schiff’s reagent for 15 min. Staining intensities were determined with ImageJ software (National Institutes of Health).

RNA Isolation and Reverse Transcription Polymerase Chain Reaction

Total RNA was isolated with TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Reverse transcription was performed with AccuPower RT PreMix (Bioneer). The resulting cDNA was amplified with AccuPower PCR PreMix (Bioneer). Primer information and polymerase chain reaction (PCR) conditions are listed in previous studies (Ishkitiev et al. 2010; Ishkitiev, Yaegaki, et al. 2012). PCR products were resolved on 1.5% agarose gels and stained with ethidium bromide.

Western Blot Analysis

Whole cell lysates and nuclear extracts were prepared. Proteins (30 µg per lane) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were then treated with blocking solution for 1 h and incubated with primary and horseradish peroxidase–conjugated secondary antibodies for 1 h at 37 °C. Proteins were visualized with enhanced chemiluminescence (Amersham).

Immunofluorescence Staining

Cells were fixed in 4% paraformaldehyde for 30 min. Primary antibodies against stage-specific embryonic antigen-4 (SSEA-4) and STRO-1 were incubated in blocking buffer for overnight at 4 °C. Alexa Fluor 488–conjugated secondary antibody (Molecular Probes) was incubated in 1% bovine serum albumin / phosphate-buffered saline for 1 h at 4 °C. For flow cytometry, 10,000 events were acquired with a FACSCalibur apparatus (BD Biosciences). Data were analyzed with CellQuest research software (BD Biosciences). Unstained cells and cells stained only with secondary antibody served as negative controls. Cells examined by confocal microscopy were mounted on glass slides and observed with a Cell Voyager CV1000 (Yokogawa Electric Corporation).

Induction of Hepatic Fibrosis and Cell Transplantation

Five-week-old BALB/c nude mice (male) were purchased from Central Lab Animal, Inc. Experimental protocols were approved by the Ethics Committee for Animal Care and Use of Kyung Hee University, Seoul, Korea. Liver fibrosis was induced by CCl₄ injection (twice weekly for 4 wk) into the peritoneum at a dosage of 1 mL/kg of body weight of CCl₄ dissolved in a 1:10 volume of castor oil (Sigma-Aldrich). Chemical marker PKH26-labeled hepatocyte-like cells derived from hDPSCs (1 × 10⁵ cells/100 µL) were transplanted into each mouse via tail vein infusion. Juglone (1 mg/kg) was injected intraperitoneally twice weekly for an additional 3 wk (Fig. 5A).

Detection of Transplanted Human Cells in Mice

To trace the transplanted hDPSCs in CCl₄-injured mice, cells were labeled with a PKH26 red fluorescent cell linker kit (Sigma-Aldrich) according to the manufacturer’s instructions and observed with a Typhoon 8600 variable mode imager (GE Healthcare). The presence of grafted human cells in the livers of CCl₄-injured mice was assessed by immunohistochemical examination. A Histostain-Plus rabbit primary kit (Zymed Laboratories, Inc.) was used to visualize immunoactions. Human cytokeratin 18 (CK18)–specific antibody was purchased from Santa Cruz Biotechnology (sc-51583).

Blood and Histologic Analysis

Mice from all experimental groups were sacrificed 7 wk after commencing the experiment. Blood was collected, and the concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and ammonia were measured with a BioVision assay kit. Specimens were fixed in 10% neutral-buffered formalin, embedded in paraffin wax via conventional techniques, sectioned at 4-μm thickness, and stained with hematoxylin and eosin. Heidenhain’s Azan trichrome staining and periodic acid–Schiff (PAS) staining were also performed to examine liver collagen deposition and glycogen storage, respectively. Fibrosis areas were measured with ImageJ 1.44 image analysis software and a CX41 microscope (Olympus) equipped with a DP21 digital camera (Olympus). The amount of collagen deposition was calculated from 25 randomized and nonoverlapping areas at 20× magnification. The percentage of fibrosis in the liver was calculated by dividing the total blue-stained area by the total liver area.

Hydroxyproline Content Assay

Hepatic hydroxyproline content in whole liver samples was quantified colorimetrically; 30 mg of freeze-dried liver samples were hydrolyzed in 6N HCl at 110 °C for 16 h. Hydroxyproline concentration was calculated from a standard curve prepared with high-purity hydroxyproline (Sigma Aldrich) and expressed as mg/g (hydroxyproline/liver).

Statistical Analysis

Comparisons of parameters among groups were made by 1-way analysis of variance combined with Bonferroni correction. All values are presented as the means ± SD. A P value <0.05 was considered statistically significant.

Results

Characterization of the Hepatic Differentiation of hDPSCs

To characterize the hepatic differentiation of hDPSCs, cells were treated with HM for up to 21 d; then their morphology and protein expression of hepatic markers were analyzed. After 21 d of culture, hDPSCs cultured in HM changed from an elongated, spindle shape to a more cuboidal or polygonal shape (Fig. 1A). Protein levels of the early hepatic marker albumin and hepatocyte nuclear factor 1α were increased, with the highest expression 5 d after HM treatment, after which levels decreased. The expression of protein levels of the mature hepatic marker CK18 was increased in a time-dependent manner, and another mature hepatic marker, CCAAT/enhancer binding protein α, was increased up to 10 d and slightly decreased thereafter (Fig. 1B). To further confirm the expression of hepatic marker in hDPSCs, hepatic differentiated hDPSCs were examined by immunocytochemistry (Fig. 1C). Intense positive staining for albumin, hepatocyte nuclear factor 1α, CK18, and CCAAT/enhancer binding protein α were detected at 21 d of HM-induced differentiated hDPSCs.

Figure 1.

Characterization of hepatic differentiation of human dental pulp stem cells (hDPSCs). Cells were treated with hepatogenic medium (HM) for 21 d. (A) Representative morphologic changes during the hepatic differentiation of hDPSCs. Scale bar = 20 µm. (B) Protein expression of hepatic markers during hepatic induction of hDPSCs. Numbers represent the relative band intensity of indicated proteins normalized to that of actin by densitometry. (C) Representative immunofluorescence images of hepatic markers in undifferentiated and differentiated hDPSCs. Scale bar = 25 µm. (D) PIN1 expression during hepatic differentiation of hDPSCs. The expression of mRNA (upper 2 bands) and protein (lower 2 bands) was assessed by reverse transcription polymerase chain reaction and Western blot analysis, respectively. These data are representative of 3 independent experiments. ALB, albumin; C/EBPα, CCAAT/enhancer binding protein α; CK18, cytokeratin 18; HNF1α, hepatocyte nuclear factors 1α.

PIN1 mRNA and Protein Expression during Hepatic Differentiation of hDPSCs

To investigate PIN1 mRNA and protein expression during hepatic differentiation of hDPSCs, cells were cultured in HM for 21 d and analyzed by conventional reverse transcription PCR and Western blotting (Fig. 1D). PIN1 mRNA and protein expression increased up until 16 h but gradually decreased thereafter. A rapid decrease in PIN1 was observed after 7 d.

Effects of PIN1 Inhibition and Overexpression on Hepatic Differentiation of hDPSCs

To examine the role of PIN1, juglone (5-hydroxy-1,4-naphthoquinone; a potent inhibitor of PIN1) was treated during the hepatic differentiation of hDPSCs. Juglone has been suggested as a reactive molecule that specifically inhibits the enzymatic activity of PIN1 (Lee et al. 2014; Costantino et al. 2016). Glycogen storage ability, which was assessed by PAS staining of hDPSC-derived hepatocyte-like cells, and expressions of hepatic marker were markedly stimulated by juglone in a dose-dependent manner (Fig. 2A, B). In Figure 2B, the relative expression of PIN1 upon juglone treatment was difficult to detect because the expression was markedly decreased after 7 d of hepatic differentiation (as shown in Fig. 1D). However, PIN1 shows a meaningful decrease in the presence of a high concentration of juglone (2 µM). STRO-1 and SSEA-4 are the best-known markers for mesenchymal stem cells (Yu et al. 2007; Kawanabe et al. 2012). Of note, pretreatment with juglone enhanced the HM-induced decrease of STRO-1- and SSEA-4-positive cells (Fig. 2C). On the contrary, PIN1 overexpression by Ad-PIN1 blocked HM-induced glycogen storage and downregulated mRNA expression of hepatic markers in hDPSCs (Fig. 2D, E).

Figure 2.

Effects of PIN1 on hepatic differentiation. To inhibit PIN1, cells were treated with juglone during the period of hepatic differentiation. Differentiation was assessed by (A) periodic acid–Schiff (PAS) staining. Bar graph shows the arbitrary PAS staining intensities. (B) Human dental pulp stem cells were treated with hepatogenic medium (HM) with indicated concentrations of juglone. The expressions of hepatic marker genes (ALB, CK18, C/EBPα and HNF1α) were examined by reverse transcription polymerase chain reaction (RT-PCR) after 10 d of culture. The expression of PIN1 was also examined by RT-PCR and Western blotting (WB). (C) Expression of stem cell surface markers was analyzed by flow cytometry (n = 4). Data are representative of 3 independent experiments. After PIN1 overexpression by infection with adenovirus (multiplicity of infection, 100) in hDPSCs, the hepatic differentiation was also assessed by (D) PAS staining and (E) RT-PCR. Adenovirus encoding β-galactosidase served as a control. Bar graph shows the arbitrary PAS staining intensities. *P < 0.05 vs. control. ^#P < 0.05 vs. HM alone. Data are representative of 3 independent experiments. Ad, adenovirus; ALB, albumin; C/EBPα, CCAAT/enhancer binding protein α; CK18, cytokeratin 18; HNF1α, hepatocyte nuclear factors 1α; LacZ, β-galactosidase.

Effects of PIN1 Inhibition and Overexpression on the Wnt3a/β-Catenin Pathway

To better understand the molecular mechanism of PIN1-mediated hepatic differentiation, we evaluated Wnt/β-catenin signaling in HM-treated hDPSCs upon PIN1 inhibition and overexpression. Among the Wnt genes examined, HM increased Wnt3a expression and subsequent total β-catenin protein expression in a time-dependent manner, whereas the expression of Wnt1 and Wnt5a remain unchanged (Fig. 3A). The presence of juglone further enhanced HM-mediated Wnt3a upregulation, while forced expression of PIN1 dramatically diminished the increase in Wnt3a (Fig. 3B). To further corroborate that PIN1 triggers a Wnt3a decrease during hepatocyte differentiation, PIN1-overexpressing hDPSCs were allowed to differentiate into hepatocytes in the presence or absence of rh-Wnt3a (Fig. 3C). In accordance with Western blot results, overexpression of PIN1 reduced the hepatocytic glycogen storage ability of hDPSCs, which was rescued by exogenous treatment with rh-Wnt3a. Similarly, restoration of hepatocyte marker genes upon Wnt3a treatment in PIN1-overexpressing cells further confirmed the significant role of PIN1 in Wnt3a reduction (Fig. 3D). In addition, DKK-1, an inhibitor of the Wnt signaling pathway, further enhanced Ad-PIN1-mediated inhibition of hepatic differentiation.

Figure 3.

Effects of PIN1 on the Wnt3a/β-catenin pathway during hepatic differentiation in human dental pulp stem cells. (A) Time-course change in the protein levels of Wnts and β-catenin. Cells were treated with hepatogenic medium (HM) at the times indicated. (B) Effects of regulating PIN1 expression on the Wnt3a/β-catenin pathway. Cells were pretreated with juglone (2 μM) for 2 h or Ad-PIN1 for 5 h and then posttreated with HM for 120 min. (C, D) Effects of rh-Wnt3a and/or Wnt inhibition (DKK1) on Ad-PIN1-supressed hepatic differentiation in human dental pulp stem cells. After the Ad-PIN1 infection for 5 h, cells were treated with HM for 7 d, with or without rh-Wnt3a (100 ng/mL) and/or DKK1 (250 ng/mL). ^#P < 0.05 vs.HM alone or HM plus Ad-LacZ; ^##P < 0.05 vs. HM plus Ad-PIN1. These data are representative of 3 independent experiments. Ad, adenovirus; ALB, albumin; C/EBPα, CCAAT/enhancer binding protein α; CK18, cytokeratin 18; HNF1α, hepatocyte nuclear factors 1α; LacZ, β-galactosidase; PAS, periodic acid–Schiff.

Confirmation of Cell Engraftment in the CCl₄-Injured Mouse Liver

To determine whether human cells were present in the livers of transplanted mice, PKH26-labeled hDPSCs were transplanted into CCl₄-injured mice (Fig. 4A). PKH26-derived fluorescence was not detected in the cryosectioned liver that did not receive transplanted cells. In contrast, significant fluorescence was observed in the liver receiving hDPSCs (Fig. 4B). The distribution of human cells in mouse liver tissue was further confirmed by immunohistochemistry for antihuman major histocompatibility complex class I (Fig. 4C). Furthermore, we performed additional immunohistochemistry using the antibody that detects human but not mouse proteins. Human-specific CK18 was positively stained in the liver tissue of the mice injected with hDPSCs (Fig. 4D). Therefore, we conclude that the differentiated hDPSCs act as hepatocytes in the murine liver after cell transplantation.

Figure 4.

Schematic diagram of an animal model for hepatic fibrosis and cell transplantation. (A) To examine engraftment of cells, prelabeled human dental pulp stem cells with PKH26 were infused into the tail vein of mice. (B, C) Representative images of incorporation of human dental pulp stem cells into carbon tetrachloride (CCl₄)–injured livers. Nontransplanted liver from CCl₄-treated mice was used as a control. While immunoreaction was absent in nontransplanted mouse livers, sections of transplanted mouse liver showed PKH26 positivity (B; original magnification, ×40) and cells expressing human major histocompatibility complex (MHC) class I (C; original magnification, ×200). (D) Representative immunohistochemical staining results for antihuman cytokeratin 18 (CK18; original magnification, ×400). Arrows indicated human CK18-positive hepatocytes in mouse liver tissue. Cytoplasmic immunoreactions against the human antibody are observed in the hepatocytes of human cell-transplanted mice.

In Vivo Therapeutic Effects of hDPSC Transplantation versus hDPSCs Plus Juglone into Mice with Liver Fibrosis

Since PIN1 inhibition by juglone promoted hepatic differentiation in hDPSCs, we next investigated the effects of juglone and hepatocyte-like cell transplantation in CCl₄-induced liver-injured mice by histologic examination and determined the serum levels of ALT, AST, and ammonia (Fig. 5A). Following 3 wk of treatment with CCl₄, liver fibrosis and destruction of sinusoidal structures were observed in sections stained with hematoxylin and eosin, PAS, and Heidenhain’s Azan trichrome (Fig. 5B). Blue-stained (fibrotic) areas decreased in mouse livers following hDPSC engraftment or juglone treatment versus treatment with CCl₄ alone. Histomorphometric examination of liver tissues showed that juglone in combination with hDPSC engraftment has a significant antifibrotic effect, as evidenced by a decrease in liver fibrosis versus the juglone-alone or hDPSC engraftment–alone group (based on Azan trichrome staining; Fig. 5C). Serum ALT, AST, and ammonia levels significantly increased in response to CCl₄ as compared with the controls but were significantly reduced in mice treated with either juglone or hDPSCs alone (Fig. 5D–F). Combined treatment of both juglone and hDPSCs significantly decreased CCl₄-induced increase of ALT, AST, and ammonia. Furthermore, hDPSC engraftment plus juglone treatment have a significant antifibrotic effect, as evidenced by the significant decrease in liver collagen (hydroxyproline content) as compared with CCl₄, CCl₄ plus juglone alone, and CCl₄ plus hDPSCs alone (Fig. 5G).

Figure 5.

Therapeutic effects of human dental pulp stem cell transplantation and juglone in carbon tetrachloride (CCl₄)–induced liver fibrosis in nude mice. (A) Schematic representation of the experimental strategy. (B) Representative histologic images of liver tissue sections from CCl₄-injured mice by hematoxylin and eosin (H&E), periodic acid–Schiff (PAS), and Heidenhain’s Azan trichrome (Azan) staining (original magnification, ×40). Arrows and arrowheads indicate areas of fibrosis. (C) Quantitative analysis of liver fibrosis in CCl₄-injured liver. Fibrotic areas were quantified in slices stained with Azan by ImageJ software. Effects of human dental pulp stem cell transplantation and juglone on serum parameters (D–F) and hepatic hydroxyproline contents (G) in mice with CCl₄-injured liver. Serum levels of alanine aminotransferase (ALT; D), aspartate aminotransferase (AST; E), and ammonia (F) were measured with an assay kit. Hepatic hydroxyproline content in whole liver samples was quantified colorimetrically (G). Values represent the means ± SD from 5 animals (C–G). *P < 0.05 vs. control; ^#P < 0.05 vs. CCl₄ only; ^##P < 0.05 vs. CCl₄ plus cell transplantation.

Discussion

Consistent with findings from previous studies, we report that hDPSCs can differentiate into cells that exhibit a hepatic phenotype in 20 to 22 d (Ishkitiev et al. 2010; Patil et al. 2014), and we verified that hDPSCs cultured in HM express high mRNA and protein levels of hepatic markers and contain PAS-positive glycogen granules. The present study also showed that PIN1 mRNA and protein expression increased in a time-dependent manner up until 16 h following hepatic induction but began to decline thereafter and was consequently suppressed for the remainder of hepatic differentiation of hDPSCs.

Wnt/β-catenin signaling is known to be a major player in the hepatocyte/liver developmental process (Thompson and Monga 2007; Monga 2014). In this study, expression levels of Wnt3a and β-catenin were enhanced in the presence of juglone but inhibited in the presence of Ad-PIN1, which is consistent with our report on the odontogenic differentiation of hDPSCs (Lee et al. 2014). In addition, the addition of rh-Wnt3a significantly reversed the inhibitory effect of Ad-PIN1on hepatic differentiation. These data support a role for PIN1 in the negative regulation of hepatocyte differentiation by inhibiting Wnt3a expression.

Engraftment is a multistep process involving directed migration of inoculated cells. The fluorescent dye PKH26, which binds mainly to the cell membrane, has been used as a cell tracer to locate transplanted cells in hosts (Li et al. 2013). In the present study, hDPSC engraftment in livers of immunocompromised mice was demonstrated by the presence of positive cells for PKH26, human major histocompatibility complex class I, and human CK18 at 3 wk posttransplantation. Our findings indicate that hDPSCs can migrate to the liver injury site and participate in liver injury healing by differentiating into hepatocytes. However, there could be another mechanism of liver injury healing by hDPSCs. Besides differentiating into hepatocytes, hDPSCs could induce hepatic differentiation of liver stem cells in the recipient mice, or hDPSCs could provide some kind of cytokines that promote liver injury healing. The precise underlying mechanism of hDPSC-mediated liver injury healing needs to be investigated in further studies.

Besides hDPSCs, several types of stem cells were evaluated as a transplantable cell sources for stem cell–based liver therapy: bone marrow–derived mesenchymal stem cells, hematopoietic stem cells, and adipose tissue–derived mesenchymal stem cells (Vosough et al. 2011; Kadyk et al. 2015). Each stem cell has its own characteristic advantages and limitations for cell-based therapy of liver repair. One of the greater advantages of DPSCs than other stem cells will be its simple and noninvasive obtaining protocol. The role of PIN1 on hepatocyte-like differentiation of other stem cells was not fully comprehended. However, recent studies report the resistance of PIN1 knockout mice to NASH (Nakatsu et al. 2012) and the attenuation of hepatocellular carcinoma upon PIN1 knockdown (Shinoda et al. 2015). These reports suggest the relevance of PIN1 to liver homeostasis, although further studies are required to understand the precise mechanisms.

Experimental liver injury by CCl₄ in nude mice is a widely used animal model to examine hepatic fibrosis by intraperitoneal administration (Cho et al. 2011). As a result of hepatic injury, liver fibrosis, the number of glycogen-laden cells, hepatic hydroxyproline contents, and serum ALT, AST, and ammonia levels showed a marked increase after CCl₄ administration; however, these increases were attenuated by hDPSCs or the PIN1 inhibitor juglone. Furthermore, the combination of hDPSCs and juglone reduced CCl₄-induced fibrosis scores. Taken together, these data demonstrate that cotreatment with hDPSC transplantation and juglone effectively reduces experimental hepatic fibrosis.

In summary, this study is the first to demonstrate that PIN1 inhibition promotes hepatic differentiation of hDPSCs via the Wnt/β-catenin pathway. Moreover, a combination therapy of hDPSC transplantation with juglone demonstrates additive effects on CCl₄-induced hepatic fibrosis. Thus, the combination of hDPSC transplantation and PIN1 inhibition may represent a potential new therapeutic approach for the treatment of hepatic fibrosis and cirrhosis.

Author Contributions

H.J. Kim, contributed to design and data interpretation, drafted the manuscript; Y.A. Cho, Y.M. Lee, S.Y. Lee, and W.J. Bae, contributed to conception and data acquisition, drafted the manuscript; E.C. Kim, contributed to conception, design, data acquisition, and analysis, drafted and critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.

Footnotes

This work was supported by a grant from the National Research Foundation of Korea, funded by the Ministry of Science, ICT and Future Planning (2012R1A5A2051384), and by a Midcareer Researcher Program grant through the National Research Foundation of Korea, funded by the Ministry of Education, Science and Technology (2012004117).

The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

References

Cho

Lim

Joo

Woo

Seoh

Cho

Han

Ryu

. 2011. Transplantation of bone marrow cells reduces CCl4 -induced liver fibrosis in mice. Liver Int. 31(7):932–939.

Cho

Jue

Bae

Heo

Shin

Kwon

Lee

Kim

. 2015. PIN1 inhibition suppresses osteoclast differentiation and inflammatory responses. J Dent Res. 94(2):371–380.

Costantino

Paneni

Lüscher

Cosentino

2016. Pin1 inhibitor juglone prevents diabetic vascular dysfunction. Int J Cardiol. 203:702–707.

de Mendonca Costa

Bueno

Martins

Kerkis

Fanganiello

Cerruti

Alonso

Passos-Bueno

. 2008. Reconstruction of large cranial defects in nonimmunosuppressed experimental design with human dental pulp stem cells. J Craniofac Surg. 19(1):204–210.

Gomes

Geraldes Monteiro

Melo

Smith

Cavenaghi Pereira da Silva

Lizier

Kerkis

Cerruti

Kerkis

2010. Corneal reconstruction with tissue-engineered cell sheets composed of human immature dental pulp stem cells. Invest Ophthalmol Vis Sci. 51(3):1408–1414.

Huang

Snyder

Cheng

Chan

. 2008. Putative dental pulp-derived stem/stromal cells promote proliferation and differentiation of endogenous neural cells in the hippocampus of mice. Stem Cells. 26(10):2654–2663.

Iohara

Zheng

Ito

Tomokiyo

Matsushita

Nakashima

2006. Side population cells isolated from porcine dental pulp tissue with self-renewal and multipotency for dentinogenesis, chondrogenesis, adipogenesis, and neurogenesis. Stem Cells. 24(11):2493–2503.

Ishkitiev

Calenic

Aoyama

Yaegaki

Imai

2012. Hydrogen sulfide increases hepatic differentiation in tooth-pulp stem cells. J Breath Res. 6(1):017103.

Ishkitiev

Yaegaki

Calenic

Nakahara

Ishikawa

Mitiev

Haapasalo

2010. Deciduous and permanent dental pulp mesenchymal cells acquire hepatic morphologic and functional features in vitro. J Endod. 36(3):469–474.

10.

Ishkitiev

Yaegaki

Imai

Tanaka

Nakahara

Ishikawa

Mitev

Haapasalo

2012. High-purity hepatic lineage differentiated from dental pulp stem cells in serum-free medium. J Endod. 38(4):475–480.

11.

Kadyk

Collins

Littman

Millan

. 2015. Proceedings: moving toward cell-based therapies for liver disease. Stem Cells Transl Med. 4(3):207–210.

12.

Kawanabe

Murata

Fukushima

Ishihara

Yanagita

Ono

Kurosaka

Kamioka

Itoh

. 2012. Stage-specific embryonic antigen-4 identifies human dental pulp stem cells. Exp Cell Res. 318(5):453–463.

13.

Kisseleva

Gigante

Brenner

. 2010. Recent advances in liver stem cell therapy. Curr Opin Gastroenterol. 26(4):395–402.

14.

Kuboki

Sakai

Clarke

Schuster

Blanchard

Edwards

Lentsch

. 2009. The peptidyl-prolyl isomerase, Pin1, facilitates NF-kappaB binding in hepatocytes and protects against hepatic ischemia/reperfusion injury. J Hepatol. 51(2):296–306.

15.

Lee

Shin

Jue

Kwon

Cho

Park

Kim

. 2014. The role of PIN1 on odontogenic and adipogenic differentiation in human dental pulp stem cells. Stem Cells Dev. 23(6):618–630.

16.

Zhang

Sun

Chen

Liu

Yao

Jiang

2013. PKH26 can transfer to host cells in vitro and vivo. Stem Cells Dev. 15(2):340–344.

17.

Zhou

. 2007. The prolyl isomerase PIN1: a pivotal new twist in phosphorylation signalling and disease. Nat Rev Mol Cell Biol. 8(11):904–916.

18.

Martens

Bronckaers

Politis

Jacobs

Lambrichts

2013. Dental stem cells and their promising role in neural regeneration: an update. Clin Oral Invest. 17(9):1969–1983.

19.

Monga

SP.

2014. Role and regulation of β-catenin signaling during physiological liver growth. Gene Expr. 16(2):51–62.

20.

Nakatsu

Otani

Sakoda

Zhang

Guo

Okubo

Kushiyama

Fujishiro

Kikuch

Fukushima

. 2012. Role of Pin1 protein in the pathogenesis of nonalcoholic steatohepatitis in a rodent model. J Biol Chem. 287(53):44526–44535.

21.

Patil

Kumar

Lee

Jeon

Jang

Lee

Park

Byun

Ahn

Kim

. 2014. Multilineage potential and proteomic profiling of human dental stem cells derived from a single donor. Exp Cell Res. 320(1):92–107.

22.

Risal

Park

Cho

Kim

Jeong

. 2012. Overexpression of peptidyl-prolyl isomerase Pin1 attenuates hepatocytes apoptosis and secondary necrosis following carbon tetrachloride-induced acute liver injury in mice. Pathol Int. 62(1):8–15.

23.

Ryo

Liou

Wulf

2003. Prolyl isomerase Pin1: a catalyst for oncogenesis and a potential therapeutic target in cancer. J Cell Sci. 116(Pt 5):773–783.

24.

Schuppan

Afdhal

. 2008. Liver cirrhosis. Lancet. 371(9615):838–851.

25.

Shinoda

Kuboki

Shimizu

Ohtsuka

Kato

Yoshitomi

Furukawa

Miyazaki

2015. Pin1 facilitates NF-κB activation and promotes tumour progression in human hepatocellular carcinoma. Br J Cancer. 113(9):1323–1331.

26.

Thompson

Monga

. 2007. WNT/beta-catenin signaling in liver health and disease. Hepatology. 45(5):1298–1305.

27.

Vosough

Moslem

Pournasr

Baharvand

2011. Cell-based therapeutics for liver disorders. Br Med Bull. 100:157–172.

28.

Wallace

Burt

Wright

. 2008. Liver fibrosis. Biochem J. 411(1):1–18.

29.

Wang

Deng

Tang

Shi

Jin

2007. Odontogenic capability: bone marrow stromal stem cells versus dental pulp stem cells. Biol Cell. 99(8):465–474.

PIN1 Suppresses the Hepatic Differentiation of Pulp Stem Cells via Wnt3a

Abstract

Keywords

Introduction

Materials and Methods

Cell Culture of hDPSCs

Preparation of Recombinant PIN1 Adenovirus

Periodic Acid–Schiff Staining

RNA Isolation and Reverse Transcription Polymerase Chain Reaction

Western Blot Analysis

Immunofluorescence Staining

Induction of Hepatic Fibrosis and Cell Transplantation

Detection of Transplanted Human Cells in Mice

Blood and Histologic Analysis

Hydroxyproline Content Assay

Statistical Analysis

Results

Characterization of the Hepatic Differentiation of hDPSCs

PIN1 mRNA and Protein Expression during Hepatic Differentiation of hDPSCs

Effects of PIN1 Inhibition and Overexpression on Hepatic Differentiation of hDPSCs

Effects of PIN1 Inhibition and Overexpression on the Wnt3a/β-Catenin Pathway

Confirmation of Cell Engraftment in the CCl4-Injured Mouse Liver

In Vivo Therapeutic Effects of hDPSC Transplantation versus hDPSCs Plus Juglone into Mice with Liver Fibrosis

Discussion

Author Contributions

Footnotes

References

Confirmation of Cell Engraftment in the CCl₄-Injured Mouse Liver