Comparative restoration of acute liver failure by menstrual blood stem cells compared with bone marrow stem cells in mice model

ARTICLE IN PRESS Cytotherapy, 2017; ■■: ■■–■■

Comparative restoration of acute liver failure by menstrual blood stem cells compared with bone marrow stem cells in mice model

MINA FATHI-KAZEROONI1, GHOLAMREZA TAVOOSIDANA1, MASOUD TAGHIZADEH-JAHED2, SAYEH KHANJANI2, HANANEH GOLSHAHI3, CAROLINE E. GARGETT4, HALEH EDALATKHAH2 & SOMAIEH KAZEMNEJAD2 1 Department of Molecular Medicine, School of Advanced Technologies in Medicine,Tehran University of Medical Sciences,Tehran, Iran, 2Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR,Tehran, Iran, 3Department of Pathology, Faculty ofVeterinary Medicine, University of Tehran,Tehran, Iran, and 4The Ritchie Centre, Hudson Institute of Medical Research, and Department of Obstetrics and Gynaecology, Monash University, Melbourne,Victoria, Australia

Abstract Background aims. The application of menstrual blood stem cells (MenSCs) in regenerative medicine is gaining increasing attention. The aim of this study was to investigate the therapeutic potential of MenSCs compared with bone marrow– derived stem cells (BMSCs) in an animal model of CCl4-induced acute hepatic failure. Methods. Injured Balb/C mice were divided into multiple groups and received MenSCs, BMSCs or hepatocyte progenitor-like (HPL) cells derived from these cells. Results. Tracking of green fluorescent protein–labeled cells showed homing of cells in injured areas of the liver. In addition, the liver engraftment of MenSCs was shown by immunofluorescence staining using anti-human mitochondrial antibody. Microscopically examination, periodic acid-Schiff and Masson’s trichrome staining of liver sections demonstrated the considerable liver regeneration post–cell therapy in all groups. Assessment of serum parameters including aspartate aminotransferase, alanine aminotransferase, total bilirubin, urea and cholesterol at day 7 exhibited significant reduction, such that this downward trend continued significantly until day 30. The restoration of liver biochemical markers, changes in mRNA levels of hepatic markers and the suppression of inflammatory markers were more significant in the MenSCtreated group compared with the BMSC-treated group. On the other hand, HPL cells in reference to undifferentiated cells had better effectiveness in the treatment of the acute liver injury. Conclusions. Our data show that MenSCs may be considered an appropriate alternative stem cell population to BMSCs for treatment of acute liver failure. Key Words: bone marrow stem cells, liver injury, menstrual blood stem cells, regenerative medicine

Introduction The adult liver has a critical role in regulating many metabolic pathways [1]. Acute liver failure (ALF) is a life-threatening condition with a high mortality rate [2]. Many patients with liver failure require a lifesaving liver transplant but are faced with major limitations including long waiting lists, lack of donors, transplant rejection and high cost [3]. In recent years, bone marrow–derived stem cells (BMSCs) are the most common type of mesenchymal stromal cells (MSCs) used in cell therapy [4–6]. Several studies showed the potential of BMSCs in regenerative medicine based on their multipotent, anti-apoptotic, immunosuppressive and paracrine properties [7,8]. Nevertheless, problems such as limited availability,

invasive sample collection and low proliferation capacity limit the applicability of BMSCs [9]. The recent identification of MSC-like cells in menstrual blood (MenSCs) provides a non-invasive source of MSCs with several advantages, such as easy accessibility without need for anaesthetic, renewability as they can be sourced on a monthly basis, high proliferative capacity in culture without inducing genetic abnormalities and lack of ethical concerns [10–13]. Moreover, immunomodulatory properties of MenSCs and their therapeutic potential in different diseases emphasize the safety and efficacy of their application for cell therapy [14–18]. Our group and others showed MenSCs can differentiate into multiple mesodermal and occasionally endodermal and ectodermal lineages [19], but with different capacities depending

Correspondence: Somaieh Kazemnejad, PhD, Tissue Engineering Department, Biotechnology Research Center, Avicenna Research Institute, ACECR, P.O. Box 19615-1177, Tehran, Iran. E-mails: [email protected], [email protected] (Received 17 March 2017; accepted 20 August 2017) ISSN 1465-3249 Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jcyt.2017.08.022

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on lineage compared with BMSCs [20–24]. More recently, in line with our project, in vivo efficiency of MenSCs in the treatment of hepatic fibrosis in the mouse model has been reported [25,26]; however, the capabilities of these cells compared with BMSCs in alleviating ALF has not been explored. We report the therapeutic potential of MenSCs compared with BMSCs in ALF by parallel biochemical, molecular and histochemical examination. We also assessed the effect of in vitro induction of differentiation into hepatocyte progenitor-like cells on the therapeutic capacity of MenSCs and BMSCs in the regeneration of liver tissue following ALF. In addition, we evaluated the immunomodulatory and antiinflammatory effects of stem cells in ALF by assessing changes in levels of inflammatory factors pre- and post-transplantation. Methods Ethics All donors of menstrual blood and bone marrow specimens signed the informed consent approved by the medical ethics committee of Avicenna Research Institute. All animals received human care in compliance with the Guide for Care and use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1985). The investigators who measured and interpreted the results of this study did not inform the design details and groups assignment. All injections and surgeries were performed by Dr. Taghizadeh, the surgeon of our study team. Isolation and culture of MenSCs and BMSCs MenSCs were separated from samples of five healthy female donors aged 20–35 years using a Diva cup during the first 2 days of the menstrual cycle. In brief, menstrual blood mononuclear cells were separated by Ficoll-Hypaque (GE-Healthcare) density gradient centrifugation. The cells were retrieved and washed, and the cell pellet was subsequently cultured in complete Dulbecco’s Modified Eagle’s Medium/F12 (Sigma-Aldrich) supplemented with 10 % fetal bovine serum (FBS), 2 mmol/L L-glutamine, 100 × non-essential amino acids, 100 U/mL penicillin, 100 mg/mL streptomycin and 25 mg/mL Fungizone and maintained at 37°C in a 5% humidified CO2 incubator. The first colonies appeared 4–7 days later. After 70–80% confluence had been reached, adherent cells were passaged using Trypsin/EDTA (Gibco). In parallel, BM-derived MSCs were obtained from bone marrow aspirates (5–10 mL) of five female donors aged 18–30 years.The specimens were aspirated from

iliac crests at the Bone Marrow Transplantation Center, Shariati Hospital, Tehran University of Medical Sciences. The isolation procedure of BMSCs was performed as described in our previous study [20,22]. Cells were used between passages 3 and 5. Characterization of MenSCs and BMSCs Evaluation of the expression of CD105 and CD146 (MSC markers), OCT-4 (embryonic stem cell marker) and CD45 (hematopoietic cell marker) was done by flow cytometric analysis as described previously [20,23]. Briefly, aliquots of 105 cells/100 µL were washed in cold phosphate buffered saline (PBS) + 2% FBS and incubated separately for 40 min with PEconjugated mouse anti-human CD105 (clone 43A3; BioLegend), CD146 (clone P1H12; BD Pharmingen) and CD45 (clone HI30; BD Pharmingen). For analysis of OCT-4 expression, the cells were washed with permeabilization buffer (0.5% saponin in PBS), treated with primary antibody, rabbit anti-human OCT-4 antibody (Abcam), for 40 min and then incubated with fluorescein isothiocyanate (FITC)-conjugated goat antirabbit immunoglobulin (Ig; Sigma). The appropriate isotype controls IgG were used as negative controls (BD Pharmingen). Finally, cells were analyzed using a Partec flow cytometer and SPSS 13 software. For further comparison of both types of MSCs, their differentiation ability into osteoblasts and adipocytes were confirmed by using alizarin red staining (Sigma-Aldrich) for detecting mineralized calcium, and oil red O staining to identify fat vacuoles, respectively. Chondrogenic differentiation was assessed by immunohistochemistry using primary monoclonal mouse anti-human collagen type II (clone 5B2.5, 1:500; Abcam) and secondary antibody FITC-labeled goat anti-mouse IgG (Abcam) using previously described protocols [20,24] provided in the online supplement. In vitro differentiation of MSCs into hepatocyte-like cells Both MenSCs and BMSCs were differentiated into hepatocyte-like cells according to the protocol reported previously [27]. In brief, cells at passage 3–4 were cultured in serum-free Dulbecco’s Modified Eagle’s Medium supplemented with 10 ng/mL basic fibroblast growth factor (b-FGF) and 20 ng/mL epidermal growth factor (EGF) for 2 days. Differentiation was induced by adding hepatogenic medium consisting of 40 ng/mL hepatocyte growth factor, 10–7 mol/l dexamethasone, 1% insulin–transferrin–selenium+1 (ITS+1), and 50 mg/mL nitrilotriacetic acid (NTA) for 14 days, followed by treatment with 10–7 mol/L dexamethasone, 1% ITS+1 and 20 ng/mL oncostatin

ARTICLE IN PRESS MenSCs and BMSCs restore liver function M for 2 weeks. The media in all steps were refreshed twice weekly. After 1 month, expression of the hepatocyte differentiation markers including cytokeratin-18 (CK18), tyrosine aminotransferase (TAT) and albumin was evaluated in both MSC types using quantitative realtime reverse transcription-polymerase chain reaction (qRT-PCR) [27]. A detailed protocol is provided in the online supplement. The induced cells were also assessed by immunofluorescent for albumin and periodic acid-Schiff (PAS) for glycogen accumulation. For immunofluorescent staining, cells were cytospin onto poly-Llysine-coated glass slides, fixed in acetone for 10 min at −20°C and were then incubated with primary antibody mouse anti-human albumin (clone HAS11, 1:100; Sigma-Aldrich) overnight at 4°C. After washing, cells were incubated for 1 h at room temperature (RT) with FITC-labeled sheep anti-mouse IgG (Avicenna Research Institute) for 45 min in the dark. As negative control, cells were stained with the mouse irrelevant IgG2a for albumin. 4′, 6-diamidino2-Phenylindole (DAPI; 1:1000; Sigma-Aldrich) staining was done to determine the nuclei of cells. The cells were examined using a fluorescence microscope (Olympus BX51) connected to a digital camera (Olympus DP71). For PAS staining, MSC seeded on poly-L-lysine mounted slides were fixed with 4% paraformaldehyde for 20 min at RT, then oxidized in 1% periodic acid for 5 min and rinsed with deionized water. Next, the treated slides with Schiff reagent (Sigma-Aldrich) rinsed with dH2O2 and subsequently nuclei were stained with Mayer’s hematoxylin for 1 min. After removing excess dye, the slides were analyzed by microscope (Olympus BX51) [27]. Green fluorescent protein transfection of MenSCs Green fluorescent protein (GFP) transfection of MenSCs was done by electroporation according to the protocol described previously [28]. Electroporation was performed using the Gene Pulser Xcell Electroporation System (Bio-Rad) at 600 V, 100 µs in a 2-mm gap cuvette. Briefly, 1 × 106 MenSCs at passage 2 resuspended in Gene Pulser Electroporation buffer (cat. no. 1652676; Bio-Rad) and were electroporated with 10 µg of linearized pEGF-N1 vector (cat. no. 6085-1; Clontech), containing the enhanced GFP and neomycin phosphotransferase genes for 100 µs. Next, transfected MenSCs were transferred to complete medium containing 10% FBS for 24 h.Then the plates were washed twice with PBS and re-fed with complete medium for 72 h to obtain stably transfected cells. Transient expression of enhanced GFP (EGFP) was about 15% (see H 5C later in the article). Finally,

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MenSCs with stable DNA integration were selected by adding 200 µg/mL G418 sulfate (cat. no. 10131027; Invitrogen) to complete medium. After selection, electroporated cells were 90% EGFP positive and the colonies of cells showed bright EGFP expression (see Figure 5C). Whole-body imaging for tracking of GFP-transduced MenSCs For cell tracking evaluation, 200 µL of 4 × 106 GFPlabeled cells/mL were transplanted into mice 48 h after CCl4 injection. After transplantation of GFP-labeled MenSCs, the mice were anesthetized via intraperitoneal injection of ketamine/xylazine (0.1 mL/20 g) to perform whole-body imaging. Animals were imaged with a Kodak in vivo imaging system FX. Bandwidths for excitation and emission of GFP were between 470 and 535 nm, respectively. Images were captured before and every 30 min until 2 h after cell transplantation. After 2 h, animals were sacrificed, and the lung, heart and liver were evaluated. Acute liver failure induction and MSCs transplantation Eight to 10-week-old female Balb/C mice (20–22 g) were housed and maintained in the animal center at the Avicenna Research Institute according to animal care guidelines. ALF in mice model was generated by intraperitoneal injection of one dose of 1 mL/kg of carbon tetrachloride (CCl4; 1:10 diluted in mineral oil). After 48 h, the injured mice were randomly divided into following groups: control group with ALF (n = 5), MenSC-treated group (n = 10), MenSC-derived hepatocyte progenitor-like (HPL) cell–treated group (n = 10), BMSC-treated group (n = 10) and BMSCderived HPL cell–treated group (n = 10). Subsequently, each mouse in treated groups received 200 µL of 4 × 106 cells/mL through the tail vein, and the control group received a transfusion of an equal volume of PBS. CCl4-untreated mice were regarded as the normal group (n = 5). Endpoint tracking of MenSC engraftment using immunohistochemistry For analysis of cell engraftment in liver tissue, 7 and 30 days after cell transplantation, liver sections from MenSC- and differentiated MenSC-treated mice were prepared for immunohistochemistry. Deparaffinized sections were rinsed with Tris-buffered saline (TBS) and blocked with 3% H2O2, then endogenous biotin was blocked with Biotin-Blocking System (cat. no. X0590; Dako) and finally blocked in mouse serum for 45 min at 4°C. To detect transplanted MenSCs, the sections were incubated overnight at 4°C with the

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mouse anti-human anti-mitochondrial antibody (MAB1273B, 1:150; Millipore). As negative control, sections stained with isotype control antibody (IgG1). After rinsing with TBS and 1% bovine serum albumin, the slides were incubated with horseradish peroxidase (HRP)-Streptavidin Conjugate (cat. no. 43– 4323, 1:150; Invitrogen) at RT for 1 h. After washing in TBS/1% bovine serum albumin, positive cells were detected using HRP-DAB Detection System (cat. no. ab64264; Abcam). Then tissue sections were washed and counter-stained with Mayer’s hematoxylin. The intensity of the tissue staining was monitored under a microscope. Post-transplantation liver function assessment At days 7 and 30 after cell transplantation, five mice of each treated groups were sacrificed to collect blood samples for biochemical analysis, and liver tissue for molecular analysis and histopathological assessment using hematoxylin and eosin (H-E), PAS and Masson’s trichrome staining. Biochemical analysis and H-E staining After centrifuging the blood, serum was collected and biochemical parameters including alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin, total bilirubin, urea and cholesterol were analyzed with the Roche Hitachi 717 chemistry analyzer. At the time of sacrifice, the animal liver tissues were fixed in 10% formalin, embedded in paraffin and cut into 5-µm-thick sections for H-E and immunohistochemical staining. PAS staining for evaluation of glycogen storage ability PAS staining was used to demonstrate intracellular glycogen in liver tissue. After cutting paraffinembedded sections at a thickness of ∼4 µm, the slides were deparaffinized and hydrated to distilled water, oxidized in 0.5% Periodic acid solution and treated as described earlier for cells. Slides were then dehydrated and cover-slipped using a synthetic mounting medium and observed under a transmitted and reflected light microscope (Olympus BX51). The glycogen was stained purple and the nuclei blue. Masson’s trichrome staining of collagen within the liver tissue For Masson’s trichrome staining, liver sections (4– 5 µm) were deparaffinized and rehydrated. After washing in distilled water, the slides were fixed in

Bouin’s solution for 1 h at 56°C and rinsed in running tap water for 5–10 min to remove the yellow color. The slides then counterstained with Weigert’s iron hematoxylin working solution for 10 min. After rinsing in running warm tap water for 10 min and washing with distilled water, slides were stained in Biebrich scarlet-acid fuchsin solution for 10–15 min. Slides were then placed in the phosphomolybdic-phosphotungstic acid solution for 10–15 min and transferred to aniline blue solution for 5–10 min. In the next step, the slides were rinsed briefly in distilled water and differentiated in 1% acetic acid solution for 2–5 min. Finally, the stained slides were dehydrated, cover-slipped and observed under the microscope (Olympus BX51) and analyzed using ImageJ software (National Institutes of Health; https://imagej.nih.gov/ij/index .html,1997–2012).

Total RNA isolation and RT-PCR Expression of hepatic markers including, albumin, CK18, cytochrome P450 family 7 Subfamily A member 1 (CYP7A1),TAT and inflammatory markers including interleukin-6 (IL-6) and cyclooxygenase-2 (COX2) at mRNA levels was examined in the samples retrieved from treated animals in all groups.Total RNA was extracted from the liver sections using AccuZol reagent according to manufacturer’s instruction. The first-strand cDNA synthesized with random primers and 1 µg DNAse-treated RNA using SuperScript II reverse transcriptase (Invitrogen), according to the manufacturer’s instructions. To examine mRNA expression, qRT-PCR was performed using ABI 7500 Real-Time PCR System as follows: initial denaturation at 95°C for 10 s, 40 cycles of a two-step PCR (95°C for 5 s, 60°C for 30 s), dissociation stage at 95°C for 15 s, 60°C for 1 min and 95°C for 15 s. β-actin was used as housekeeping gene to normalize expression data. The primer sequences are listed in Table I.

Statistical analysis The data are presented as the mean ± SD. The quantitative results were analyzed by SPSS 13 statistical software. Comparisons between groups were done using analysis of variance with multiple comparisons post hoc test in normally distributed quantitative variables. A P value <0.05 denoted a statistically significant difference. Survival duration curves were plotted according to the method of Kaplan and Meier [29] and compared between groups by the log-rank test. Mean efficiencies and crossing point values for each gene was determined with LinRegPCR

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Table I. Sequences of the primers used in the qRT-PCR analysis. Gene of interest ALB CK-18 CYP7A1 TAT IL-6 COX-2 B-Actin

Sequence

Product size (bp)

Forward: 5′-AAGGCTACAGCGGAGCAAC-3′ Reverse: 5′-GACAAGGTTTGGACCCTCAGTC-3′ Forward: 5’-GTGAAGAGCCTGGAAACTGAGA-3′ Reverse: 5′-CATCTACCACCTTGCGGAGT-3′ Forward: 5′-ACAACGGGTTGATTCCATACC-3′ Reverse: 5′-GTCCAAATGCCTTCGCAGA-3′ Forward: 5′-CGCTTCCTATTACCACTGTCC-3′ Reverse: 5′-ACTCAGCCAATGTCCTGTAGA-3′ Forward: 5′-CACGGCCTTCCCTACTTCAC-3′ Reverse: 5′-GCAAGTGCATCATCGTTGT-3′ Forward: 5′-CAGCACTTCACCCATCAGT-3′ Forward: 5′-GATACACCTCTCCACCAATG-3′ Forward: 5′-GTCGAGTCGCGTCCACC-3′ Reverse: 5′-CATTCCCACCATCACACCCTG-3′

117 283 228 167 186 181 114

ALB, albumin; CK-18, cytokeratin-18; CYP7A1, cytochrome P450 7A1; TAT, tyrosine aminotransferase; IL-6, interleukin 6; COX-2, cyclooxygenase 2.

(version 11.0) using relative expression software tool-2009 (REST-2009; available at http://www.genequantification.de/rest-2009.html) [30]. Results In vitro characterization of MenSCs and BMSCs Similar to BMSCs, MenSCs had spindle-shaped morphology in culture. However, MenSCs colonies were smaller and more compact than those of BMSCs (Figure 1A). Both MSC types were positive for CD105 and CD146 and negative for hematopoietic marker CD45. In contrast, the expression of OCT-4 as the embryonic marker was only positive in MenSCs (Figure 1B). As shown in Figure 1C, MenSCs differentiated into mesodermal lineages as shown by collagen II-expressing chondrocyte-like cells and mineralized calcium producing osteoblasts in a similar manner with BMSCs. However, BMSCs showed a greater potential than MenSCs for differentiation into adipocytes.

Establishment of animal model with ALF and cell density for transplantation To induce acute liver failure in mice, three doses of undiluted CCl4 (0.5, 1 and 1.5 ml/kg) were injected intraperitoneally and mice were evaluated for appearance, activity, serum liver enzyme and liver pathological changes (Figure 3A). A dose of <1 mL/kg body weight did not induce sufficient hepatotoxicity, whereas a dose >1 mL/kg body weight resulted in very high mortality rate due to fulminant hepatic failure. Therefore, the dose of 1 mL/kg body weight of 10% CCl4 was selected for following experiments (Figure 3A). Necropsy of CCl4-treated mice showed ascites, peritoneal adhesion and a severe edematous pale liver with multiple nodules (Figure 4). Several different doses of cells (6 × 106, 5 × 106, 4 × 106 and 2.5 × 106 per mL) in 200 µL PBS was injected via tail vein. As determined by survival rate, the density of 4 × 106/mL was safe and efficient for injection.The animals in control CCl4-treated group died within 60 h post-CCl4 injection, whereas 70–80% of the MenSCs, BMSC- and HPL-treated mice survived for prolonged periods (Figure 3B).

Evaluation of differentiated cells into hepatocyte-like cells After 30 days of differentiation, the fibroblastic-like shape of MenSCs and BMSCs changed to a flattened and triangular epithelial morphology (Figure 1A). Real-time PCR data showed that both differentiated BMSCs and MenSCs expressed similar levels of albumin; however, the expression levels of CK18 and TAT were higher in MenSCs compared with BMSCs (Figure 2A). In addition, differentiated cells from both MSC types expressed albumin protein judged by immunofluorescent staining (Figure 2B) and were positive for PAS staining (Figure 2C).

GFP-transduced MenSCs migrated into the injured liver To localize GFP-labeled MenSCs in animals receiving cell transplantation, images were captured every 30 min. As shown in Figure 5 (A,B), we demonstrated distribution of GFP-positive MenSCs in the mouse lung and liver after 30 min. GFP-positive cells were recognized as tiny spots expressing green fluorescence above the background. The signal intensity steadily increased from 30 min to 120 min. Over 2 h, accumulation of injected GFP-positive cells was

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Figure 1. (A) Morphology of MenSCs, BMSCs and their differentiated derivatives into HPL cells. Scale bar: 100 µm. (B) Representative histograms of both MSCs immunophenotyping by flow cytometry. CD markers are demonstrated in red and the respective isotype control is shown in black. The results are representative of three to five independent experiments. (C) MenSCs and BMSCs differentiation into chondrocytes (1), adipocytes (2) and osteoblasts (3), judged by immunostaining of collagen type II, oil red O staining and alizarin red staining. Scale bar: 100 µm.

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Figure 2. Histological and molecular evaluation of differentiated MenSCs and BMSCs. (A) qRT-PCR liver markers in differentiated cells normalized to corresponding glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and calculated with reference to undifferentiated cells. Data are mean ± SD of five independent samples (*P < 0.05). (B) Albumin in differentiated cells was examined by immunofluorescence with DAPI nuclear staining. Scale bar: 100 µm. (C) PAS staining of glycogen storage in undifferentiated and differentiated cells. Scale bar: 100 µm.

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Figure 3. (A) Kaplan-Meier survival curves for mice treated with different doses of CCl4 injection. (B) On the basis of survival rate, a dosage of 4 × 106/ml was chosen as the optimal dosage for cell injection via tail vein. (C) MSC therapy significantly prolonged the survival of mice compared with CCl4-treated animals.

observed densely around the injured foci of the liver and lung (Figure 5A,B). MenSCs and HPL cells engraft into the damaged host livers Liver tissue regeneration following MSC transplantation depends on MenSCs homing to the injury site. Injection of MenSCs into the systemic circulation resulted in homing of these cells to foci of injury, the first important step for the liver injury repair.

Immunohistochemistry staining of liver sections using anti-human mitochondrial antibody showed that MenSCs and HPL cells appeared to engraft into the portal areas of host liver 1 and 4 weeks after transplantation (Figure 5D). Both MenSCs and BMSCs improved the biochemical function of transplanted livers Serum levels of AST, ALT, total bilirubin, urea and cholesterol were significantly higher 48 h after CCl4

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Figure 4. (A) Gross morphology and H-E staining of CCl4-damaged livers treated with and without MenSCs, BMSCs and HPL cells. Yellow arrows show areas with fibrosis in the livers. The images are representative of sections from five mice in each group. Scale bar: 100 µm.

treatment compared with the normal group (P < 0.01), although this was not significant for albumin (Figure 6). The average elevation in serum levels of AST, ALT total bilirubin, cholesterol and urea were 9.1-, 18.8-, 2.1-, 1.5- and 1.4-fold, respectively. Seven days after MenSC and BMSC infusion, the levels of AST, ALT, cholesterol, total bilirubin and urea were decreased significantly and continued to trend down during the next month compared with respective values of the CCl4treated controls (P < 0.05). Biochemical analysis indicated that 7 days after MenSC and BMSC transplantation, AST level was decreased by 85.9% and 81.1%, respectively (P < 0.05); similarly, transplantation

of MenSC- and BMSC-derived HPL cells resulted in a decrease in AST level of 86.5% and 81.9%, respectively (P < 0.05, Figure 6). MenSC transplantation also had a greater effect in reducing ALT compared with BMSC transplantation (87.1% vs. 82.2%, P < 0.05) at 7 days. Furthermore, HPL cells derived from MenSCs and BMSCs resulted in significant decreases of ALT levels compared with undifferentiated forms (P < 0.05). Serum level of total bilirubin was significantly reduced 7 days after transplantation with both types of MSC; however, MenSCs and MenSC-derived HPL cells were more effective than BMSCs and BMSC-derived HPL cells in

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Figure 5. Tracking and engraftment of transplanted MenSCs into CCl4-damaged host liver. (A) After intravenous injection of GFPlabeled MenSCs, images were captured at time 0, and every 30 min thereafter, Images are representative of five animals. Over 2 h, accumulation of injected GFP-positive cells (black arrows) was observed densely around the injured foci of the liver and in the lungs. (B) After sacrificing, large numbers of GFP-expressing cells (arrow) were detected in the inflamed areas of liver, lung and heart. (C) Expression of EGFP in MenSCs (1) negative control, (2) 3 days after electroporation (3) and stable expression of EGFP in a clone of MenSCs, 3 weeks after selection in G418. Scale bar: 100 µm. (D) Cells stained with human mitochondrial protein (arrow) in liver samples retrieved from animals treated with MenSC- and MenSC-derived HPL cells, 1 and 4 weeks after transplantation. Liver section from normal mice served as negative control. Scale bar: 100 µm.

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Figure 6. Improvement of hepatic function at days 7 and 30 after cell transplantation. The levels of AST, ALT, albumin (ALB) total bilirubin (T.Bili) urea and cholesterol (Chol), were measured in samples retrieved from five animals in each group. *Significant difference between CCl4-treated group and normal (NL) mice (without treatment). **Significant difference between cell therapy groups and CCl4treated group (P < 0.05). +Significant difference between two cell therapy groups at the same time.

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reducing of total bilirubin (P < 0.05). Furthermore, the similar and significant trends in reduction of these biochemical liver markers were observed after 30 days. The serum level of urea showed a notable decrease in all cell-treated groups 7 days after cell therapy, and the differentiated MenSCs reduced urea levels more than did differentiated BMSCs at both time points (P < 0.05). Finally, there were significant progressive decreases in cholesterol levels of all treated groups at day 7, and the level of this liver marker decreased to baseline at 30 days after MSC transplantation (P < 0.05), but no significant differences were observed between different groups at either 7 or 30 days (Figure 6). Cell transplantation decreased hepatocyte degeneration and inflammatory cell infiltration H-E staining of CCl4-treated livers showed the multifocal areas of coagulation necrosis in centrilobular and periportal areas. In addition vacuolar change, hydropic degeneration of hepatocytes, infiltration of inflammatory cells and activation of Kupffer cells were seen in areas of injury (Figure 4). Microscopic examination of liver sections at day 7 showed that transplantation of both MSC types limited the tissue damage compared with the CCl4treated group. Only minor derangement of the hepatocyte cords, portal inflammation, slight bile duct hyperplasia, vascular congestion with some small foci of necrotic cells and hemorrhage were also observed (Figure 4). After 30 days, there was a clear improvement in the histopathological appearance of the livers of MenSCs and BMSC-transplanted groups. Microscopic examination of the MenSC-derived HPL cell– treated group compared with the BMSC-derived HPL cell–treated group exhibited greater hepatic recovery characterized by a relatively satisfactory regeneration of hepatocytes and fewer inflammatory cells around the portal areas (Figure 4). Improvement in glycogen storage ability of the injured liver after cell transplantation PAS staining of CCL4-treated liver sections showed severe depletion of mucopolysaccharides in areas with coagulative necrosis. After 1 week, in the MenSC- and MenSC-derived HPL cell–treated groups, notable increases in intensity of the positive reaction of the stain were observed, whereas BMSC- and BMSC-derived HPL cell–treated groups demonstrated partial improvement in mucopolysaccharides content of hepatocytes (Figure 7). At day 30, the MenSC group showed moderate positivity to the stain over the hepatic lobules; however, the MenSC-derived HPL cell– treated group demonstrated a noticeable improvement

in mucopolysaccharides content (Figure 7). In the BMSC- and BMSC-derived HPL cell–treated groups, increases in the positivity of cells to PAS staining were observed compared with CCl4 group (Figure 7).These data demonstrate that both types of stromal cells improve the glycogen storage ability of host livers after CCl4 injury, especially the MenSC-derived HPL cell– treated group. Decrease in collagen fiber deposition in liver sections after MenSCs transplantation As expected, CCl4 led to liver damage, indicated by pronounced morphological alterations and disruption of the tissue architecture. Seven days after cell transplantation, gross inspection of dissected liver tissues showed spot necrosis of the liver parenchyma (Figure 4), and microscopic liver morphology showed excessive accumulation of collagen in Masson’s trichrome–stained sections in all treated groups (Figure 7). However, after 30 days, gross examination of liver tissues taken from the BMSC- and differentiated BMSC-treated groups appear to show more fibrotic areas compared with the MenSC- and differentiated MenSC-treated groups (Figure 4). Furthermore, photomicrography of liver sections showed more areas of bridging collagen fiber accumulation with large portal-portal fibrous septa formation in the BMSC- and BMSC-derived HPL cell–treated groups (38.44 ± 5.34% and 29.56 ± 3.12%, respectively) compared with the MenSC- and MenSC- derived HPL cell–treated groups (24.84 ± 6.15% and 17.45 ± 8.62%, respectively; P < 0.01; Figure 7). Hepatic and inflammatory genes expression after stem cell transplantation To further assess the reparative effects of MenSCs and BMSCs and their HPL derivatives, we assessed hepatic and inflammatory gene expression. The mRNA expression level of the liver-specific marker albumin decreased significantly after CCl4 therapy (P < 0.05). Seven days after cell therapy, albumin gene expression level was up-regulated in all groups (P < 0.05; Figure 8). This trend continued during the treatment period for all cell-treated groups; the MenSCderived HPL cell–treated group had the maximum upregulation of albumin gene expression after 30 days (P < 0.05). Similarly, CK-18 mRNA markedly reduced after CCl4 and 7 days post–cell transplantation, a significant up-regulation of the CK-18 gene was observed in MenSC- and BMSC-derived HPL cell–transplanted groups compared with MenSC- and BMSCtransplanted groups, respectively (P < 0.05) (Figure 8). At 30 days after cell transplantation, the expression of CK-18 exhibited significant up-regulation in the MenSC-derived HPL cell–treated group compared

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Figure 7. PAS and Masson’s trichrome staining of livers retrieved from animals treated with CCl4, MenSCs, BMSCs and HPL cells 7 and 30 days post-transplantation. The images are representative of sections from five mice in each group. Scale bar: 100 µm.

with the other groups (P < 0.05). In addition, expression of CYP7A1 and TAT genes at the mRNA level were up-regulated 7 days post–cell transplantation in all groups (P < 0.05). Also, comparison between the MenSC- and BMSC-treated groups showed that the MenSC- and MenSC-derived HPL cell–transplanted groups had significantly greater expression compared with the BMSC- and BMSC-derived HPL cell– treated groups, respectively (P < 0.05). After 30 days, significant up-regulation of CYP7A1and TAT genes remained in MenSC-derived HPL cell–treated group compared with BMSC-derived HPL cells (P < 0.05; Figure 8). Gene expression of the inflammatory markers, COX-2 and IL-6 increased after CCl4 treatment and following MSC therapy these were suppressed (Figure 7). After 30 days, MenSC- and MenSCderived HPL cells had greater suppressive effect on

COX-2 and IL-6 gene expression than BMSCs and BMSC-derived HPL cells (P < 0.01; Figure 8). Discussion The key findings of this study were that MenSCs and their derivative HPL cells showed greater reparative effect in an acute liver failure mouse model than did BMSCs and BMSC-derived HPL cells judged by biochemical, molecular and histopathological analysis.The greater suppression of inflammatory markers posttransplantation of MenSCs compared with BMSCs may indicate stronger immunomodulatory properties of MenSCs. Similarly, several biochemical markers of liver injury (AST, ALT, total bilirubin) showed that MenSCs exerted greater tissue reparative activity compared with BMSCs. Moreover, our study showed that in vitro induction of both types of MSC into hepatocyte

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Figure 8. qRT-PCR results of hepatic and inflammatory genes post–cell transplantation. Data of different groups were normalized to corresponding β-actin and calculated in reference to control normal (NL) group. +*Significance between cell treatment groups at different interval times. **Significance between MenSC-treated groups compared with BMSC-treated groups in the similar situation. All parameters in different groups showed significant changes after cell therapy compared with CCl4-treated group. ++Significance between groups treated with undifferentiated and differentiated cells from the similar source and the same evaluation time (P < 0.05). Data are mean ± SD of n = 5 animals/group.

ARTICLE IN PRESS MenSCs and BMSCs restore liver function progenitor cells could regenerate ALF with higher capacity compared with undifferentiated cells. Monitoring of implanted GFP-labeled MenSCs demonstrated migration and engraftment of MenSCs into injured areas of the host’s liver. ALF is a critical condition characterized by severe liver injury and hepatic encephalopathy with poor prognosis [31]. BMSCs therapy, characterized by high cell plasticity, anti-inflammatory, immunomodulatory and trophic effects, has been proposed as an alternative treatment for orthotopic liver transplantation [32]. In our previous studies, we have demonstrated the ability to isolate MSCs from menstrual blood samples and similarity of MenSC and BMSC characteristics. Despite this similarity, MenSCs have some properties that distinguish them from BMSCs, including their ability to trans-differentiate into various endodermal lineages including hepatocytes, presenting different patterns of hepatocyte markers compared with BMSCs [24,27]. These differences between MenSCs and BMSCs led us to examine the therapeutic efficacy of MenSCs compared BMSCs in an ALF animal model. We established an ALF mice model by intraperitoneal injection of CCl4, the most common hepatotoxin used to induce oxidative stress and centrilobular necrosis in rodents [33]. It is notable that a slight migration of GFP-labeled MenSCs arose in the lung in addition to the liver, which could be attributed to an intoxication effect of CCl4 due to the generation of free reactive oxygen radicals [34]. The pathological changes in pulmonary circulation in mice with liver failure have also been reported in previous studies [35].This may provide a useful tool for future studies of the molecular mechanisms mediating the pulmonary vascular diseases associated with liver failures. Consistent with previous studies, we observed gross pathological changes and aggregation of inflammatory cells in CCl4-treated animals, typical of severe ALF, with death of all untreated animals within 2 days [36,37]. At the same time, cell therapy using MenSCs and BMSCs (undifferentiated or differentiated) significantly improved the survival of mice in this ALF model. In contrast to BMSC-treated groups, the survival rate of mice treated with MenSC- derived HPL cells was better than that of undifferentiated MenSCs. However, in both stem cell types, the differences were not significant. Similar to other studies, the current study showed restoration of the gross appearance of treated livers, which were very similar to normal animals [32,38]. In corroboration with this assertion, cell transplantation in all groups significantly alleviated hepatocyte vacuolization, infiltration of inflammatory cells and vascular congestion. These results were confirmed by

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biochemical, histopathological and molecular analysis 7 and 30 days after cell transplantation. In this study, we demonstrated that MenSCs had greater restorative capacity in ALF than BMSCs, which was evident at 7 days; this trend continued until day 30, particularly for the liver enzymes and hepatocyte molecular markers. MenSC transplantation was even more effective than treatment with BMSCs in suppressing inflammation, as determined by liver tissue levels of COX-2 and IL-6 mRNA. Moreover, pathological assessment of PAS staining demonstrated a noticeable improvement in the mucopolysaccharide content of liver tissues in the MenSCs group compared with the BMSCs group. In line with these differences, Masson’s trichrome staining of liver tissues showed greater collagen fiber accumulation as large fibrous septa in the BMSC-treated group compared with that in the MenSC-treated group. The upregulation of hepatic-specific markers after cell transplantation is further evidence of the restoration of liver function in ALF [39]. On the basis of our results, mRNA expression of hepatic markers exhibited a variable pattern in MenSCand BMSC-treated groups. Notably, a significant improvement in liver function tests (i.e., CPY7A1,TAT) was observed in both the differentiated or undifferentiated MenSC-treated groups compared with the differentiated or undifferentiated BMSC-treated groups 7 and 30 days after cell transplantation. Consistent with these results, others have reported the relative effectiveness of various MSCs sourced from amniotic fluid, adipose tissue and bone marrow in the treatment of ALF [36–38,40], but our study is the first to show that MenSCs, the most accessible stem cell population, have more reparative properties for ALF compared with BMSCs. To clarify whether similar restoration can be mediated by undifferentiated and differentiated cells, we studied the effectiveness of transplanted MenSC- and BMSC-derived HPL cells. Similar to previous studies that assessed differentiated amniotic fluid- and adipose tissue-derived MSCs [36,41,42], our results also indicated that the reduction of liver enzymes, restoration of liver architecture and suppression of inflammatory markers was significantly different between the undifferentiated and differentiated cells. Differentiated HPL cells derived from both MenSCs and BMSCs were superior to their undifferentiated counterparts in all of the studied parameters. Furthermore, our study showed promising results for MenSC-derived HPL cells, which had greater restorative capacity than BMSCderived HPL cells in the treatment of ALF. Although the mechanism through which MSC therapy mediates liver regeneration has not yet been clarified in previous studies, two possible mechanisms may be involved: (i) cell migration into the injured

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areas and differentiation into hepatocyte-like cells or (ii) trophic effects through the secretion of various growth factors and cytokines that exert a paracrine effect on the behavior of resident cells [43,44]. We also demonstrated successful liver engraftment of MenSCs and HPL cells in ALF model 7 days after transplantation. The engraftment of both MenSCs and their HPL derivatives in the injured liver was demonstrated by anti-human mitochondrial antibody for up to 1 month post-infusion, providing assurance of cell engraftment and survival for up to 30 days post-transplantation. One common mechanism by which MSCs improve damaged tissue is through their immunomodulatory effects. Our data demonstrate that both MenSCs and BMSCs had immunomodulatory effects by downregulating COX-2 and IL-6 gene expression, two important molecules involved in initiation and progression of liver injury [45–47]. IL-6 hyperstimulation appears to be one of the main causes of liver impairment [46]. IL-6 is also considered an inflammatory biomarker because of its roles in with inflammation and cell proliferation [47]. Our current findings indicated that after 1 and 4 weeks, a greater suppression of IL-6 was observed in the MenSC-treated group compared with the BMSC-treated group, as well as a notable down-regulation of COX-2 mRNA in the MenSC-treated group. In line with our study, a recent study undertaken concurrently with ours showed that treatment of liver injury in a mouse model with menstrual blood–derived endometrial regenerative cells dramatically reduced the levels of pro-inflammatory cytokines, including IL-6 [25]. In summary, we have demonstrated the effectiveness of MenSCs in the restoration of ALF in a mouse model with higher capability than classic BMSCs across a broad range of biochemical and molecular markers in regenerating acute liver damage. Because BMSCs have limitations, such as invasive sampling and the agerelated decreases in number and plasticity, MenSCs provide a new, easily obtained source of MSCs, with promising therapeutic potential for liver failure. MenSCderived HPL cells showed great ability to engraft into the injured liver, suppress the inflammatory process and thus improve liver function tests. In the future, more studies on possible mechanisms underlying the effect of MenSCs on liver tissue restoration should be defined with comparisons between BMSCs and other tissue sources of MSCs.These preclinical studies underscore the potential utility of MenSCs, derived from waste tissue, which could provide an MSC therapy for a variety of diseases associated with acute liver damage and possibly for other more chronic liver disorders. However, the evaluation of MenSCs in standardized clinical trials is required to prove this assertion and determine their future application in the clinic.

Acknowledgments The authors thank Dr. Amir-Hassan Zarnani (Avicenna Research Institute) for his technical assistance in immunohistochemistry. This work was supported by a grant from Tehran University of Medical Sciences (TUMS; grant 9302-87-25918) and Avicenna Research Institute (grant 92-1158). Disclosure of interest: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article. References [1] Ghany MG, Hoofnagle JH. Approach to the patient with liver disease. In: Kasper D, Fauci A, Hauser S, Longo D, Jameson J, Loscalzo J, editors. Harrison’s principles of internal medicine. 19th ed. New York: McGraw-Hill; 2015. p. 1989–95. [2] McPhail MJ, Kriese S, Heneghan MA. Current management of acute liver failure. Curr Opin Gastroenterol 2015;31:209– 14. [3] Zarrinpar A, Busuttil RW. Liver transplantation: past, present and future. Nat Rev Gastroenterol Hepatol 2013;10:434–40. [4] Wei X, Yang X, Han Z-P, Qu F-F, Shao L, Shi Y-F. Mesenchymal stem cells: a new trend for cell therapy. Acta Pharmacol Sin 2013;34:747–54. [5] U.S. National Institutes of Health. Study Protocol of Intramyocardial Injection of Autologous Bone Marrow Stem Cells for Refractory Angina (ReACT); 2013. Available from: http://clinicaltrials.gov/ct2/show/NCT01966042. [Accessed 14 October 2013]. [6] U.S. National Institutes of Health. Treatment of Knee Osteoarthritis With Autologous Mesenchymal Stem Cells (KDD & MSV); 2010. Available from: http://clinicaltrials .gov/ct2/show/NCT01183728. [Accessed 13 August 2010]. [7] Krampera M, Glennie S, Dyson J, Scott D, Laylor R, Simpson E, et al. Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide. Blood 2003;101:3722–9. [8] Le Blanc K, Ringden O. Immunomodulation by mesenchymal stem cells and clinical experience. J Intern Med 2007;262: 509–25. [9] Ullah I, Subbarao RB, Rho GJ. Human mesenchymal stem cells-current trends and future prospective. Biosci Rep 2015;35:e00191. [10] Allickson J, Xiang C. Human adult stem cells from menstrual blood and endometrial tissue. J Zhejiang Univ Sci B 2012; 13:419–20. [11] Patel AN, Park E, Kuzman M, Benetti F, Silva FJ, Allickson JG. Multipotent menstrual blood stromal stem cells: isolation, characterization, and differentiation. Cell Transplant 2008;17:303–11. [12] Ulrich D, Muralitharan R, Gargett CE. Toward the use of endometrial and menstrual blood mesenchymal stem cells for cell-based therapies. Expert Opin Biol Ther 2013;13:1387– 400. [13] Darzi S, Werkmeister JA, Deane JA, Gargett CE. Identification and characterization of human endometrial mesenchymal stem/stromal cells and their potential for cellular therapy. Stem Cells Transl Med 2016;5:1127–32. [14] Luz-Crawford P, Torres MJ, Noël D, Fernandez A, Toupet K, Alcayaga-Miranda F, et al. The immunosuppressive signature of menstrual blood mesenchymal stem cells entails

ARTICLE IN PRESS MenSCs and BMSCs restore liver function

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29] [30]

opposite effects on experimental arthritis and graft versus host diseases. Stem Cells 2016;34:456–69. Nikoo S, Ebtekar M, Jeddi-Tehrani M, Shervin A, Bozorgmehr M, Kazemnejad S, et al. Effect of menstrual blood-derived stromal stem cells on proliferative capacity of peripheral blood mononuclear cells in allogeneic mixed lymphocyte reaction. J Obstet Gynaecol Res 2012;38:804–9. Borlongan CV, Kaneko Y, Maki M, Yu S-J, Ali M, Allickson JG, et al. Menstrual blood cells display stem cell-like phenotypic markers and exert neuroprotection following transplantation in experimental stroke. Stem Cells Dev 2010;19:439–52. Rodrigues MCO, Dmitriev D, Rodrigues A Jr, Glover LE, Sanberg PR, Allickson JG, et al. Menstrual blood transplantation for ischemic stroke: therapeutic mechanisms and practical issues. Interv Med Appl Sci 2012;4:59–68. Liu T, Huang Y, Zhang J, Qin W, Chi H, Chen J, et al. Transplantation of human menstrual blood stem cells to treat premature ovarian failure in mouse model. Stem Cells Dev 2014;23:1548–57. Gargett CE, Schwab KE, Deane JA. Endometrial stem/ progenitor cells: the first 10 years. Hum Reprod Update 2015;22:137–63. Darzi S, Zarnani AH, Jeddi-Tehrani M, Entezami K, Mirzadegan E, Akhondi MM, et al. Osteogenic differentiation of stem cells derived from menstrual blood versus bone marrow in the presence of human platelet releasate. Tissue Eng Part A 2012;18:1720–8. Lin J, Xiang D, Zhang JL, Allickson J, Xiang C. Plasticity of human menstrual blood stem cells derived from the endometrium. J Zhejiang Univ Sci B 2011;12:372–80. Kazemnejad S, Akhondi MM, Soleimani M, Zarnani AH, Khanmohammadi M, Darzi S, et al. Characterization and chondrogenic differentiation of menstrual blood-derived stem cells on a nanofibrous scaffold. Int J Artif Organs 2012;35:55– 66. Khanmohammadi M, Khanjani S, Edalatkhah H, Zarnani A, Heidari-Vala H, Soleimani M, et al. Modified protocol for improvement of differentiation potential of menstrual bloodderived stem cells into adipogenic lineage. Cell Prolif 2014;47:615–23. Khanjani S, Khanmohammadi M, Zarnani AH, Talebi S, Edalatkhah H, Eghtesad S, et al. Efficient generation of functional hepatocyte-like cells from menstrual blood-derived stem cells. J Tissue Eng Regen Med 2015;9:124–34. Chen L, Zhang C, Chen L, Wang X, Xiang B, Wu X, et al. Human menstrual blood-derived stem cells ameliorate liver fibrosis in mice by targeting hepatic stellate cells via paracrine mediators. Stem Cells Transl Med 2016;6:272–84. Lu S, Shi G, Xu X, Wang G, Lan X, Sun P, et al. Human endometrial regenerative cells alleviate carbon tetrachlorideinduced acute liver injury in mice. J Transl Med 2016;14:300. Khanjani S, Khanmohammadi M, Zarnani AH, Akhondi MM, Ahani A, Ghaempanah Z, et al. Comparative evaluation of differentiation potential of menstrual blood-versus bone marrow-derived stem cells into hepatocyte-like cells. PLoS ONE 2014;9:e86075. Peister A, Mellad J, Wang M, Tucker H, Prockop D. Stable transfection of MSCs by electroporation. Gene Ther 2004;11:224–8. Kaplan EL, Meier P. Nonparametric estimation from incomplete observations. J Am Stat Assoc 1958;53:457–81. Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 2002;30:e36.

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[31] Patton H, Misel M, Gish RG. Acute liver failure in adults: an evidence-based management protocol for clinicians. Gastroenterol Hepatol 2012;8:161–212. [32] Berardis S, Sattwika PD, Najimi M, Sokal E. Use of mesenchymal stem cells to treat liver fibrosis: current situation and future prospects. World J Gastroenterol 2015;21:742–58. [33] Weber LW, Boll M, Stampfl A. Hepatotoxicity and mechanism of action of haloalkanes: carbon tetrachloride as a toxicological model. Crit Rev Toxicol 2003;33:105–36. [34] Marques TG, Chaib E, Fonseca JHD, Lourenço ACR, Silva FD, Ribeiro MAF Jr, et al. Review of experimental models for inducing hepatic cirrhosis by bile duct ligation and carbon tetrachloride injection. Acta Cir Bras 2012;27:589–94. [35] Das M, Boerma M, Goree JR, Lavoie EG, Fausther M, Gubrij IB, et al. Pathological changes in pulmonary circulation in carbon tetrachloride (CCl4)-induced cirrhotic mice. PLoS ONE 2014;9:e96043. [36] Zagoura DS, Roubelakis MG, Bitsika V, Trohatou O, Pappa KI, Kapelouzou A, et al. Therapeutic potential of a distinct population of human amniotic fluid mesenchymal stem cells and their secreted molecules in mice with acute hepatic failure. Gut 2012;61:894–906. [37] Yin L, Zhu Y, Yang J, Ni Y, Zhou Z, Chen Y, et al. Adipose tissue-derived mesenchymal stem cells differentiated into hepatocyte-like cells in vivo and in vitro. Mol Med Rep 2015;11:1722–32. [38] Itaba N, Matsumi Y, Okinaka K, Ashla AA, Kono Y, Osaki M, et al. Human mesenchymal stem cell-engineered hepatic cell sheets accelerate liver regeneration in mice. Sci Rep 2015;5:16169. [39] Raafat N, Aal SMA, Abdo FK, El Ghonaimy NM. Mesenchymal stem cells: in vivo therapeutic application ameliorates carbon tetrachloride induced liver fibrosis in rats. Int J Biochem Cell Biol 2015;68:109–18. [40] Huang B, Cheng X, Wang H, Huang W, Wang D, Zhang K, et al. Mesenchymal stem cells and their secreted molecules predominantly ameliorate fulminant hepatic failure and chronic liver fibrosis in mice respectively. J Transl Med 2016;14:45. [41] Winkler S, Hempel M, Brückner S, Mallek F, Weise A, Liehr T, et al. Mouse white adipose tissue-derived mesenchymal stem cells gain pericentral and periportal hepatocyte features after differentiation in vitro, which are preserved in vivo after hepatic transplantation. Acta Physiol 2015;215:89–104. [42] Aurich I, Mueller LP, Aurich H, Luetzkendorf J, Tisljar K, Dollinger MM, et al. Functional integration of hepatocytes derived from human mesenchymal stem cells into mouse livers. Gut 2007;56:405–15. [43] Liu WH, Song FQ, Ren LN, Guo WQ, Wang T, Feng YX, et al. The multiple functional roles of mesenchymal stem cells in participating in treating liver diseases. J Cell Mol Med 2015;19:511–20. [44] Eom YW, Shim KY, Baik SK. Mesenchymal stem cell therapy for liver fibrosis. Korean J Intern Med 2015;30:580–9. [45] Yu J, Ip E, Hou JY, Sesha J, Pera N, Hall P, et al. COX-2 induction in mice with experimental nutritional steatohepatitis: role as pro-inflammatory mediator. Hepatology 2006;43:826– 36. [46] Campbell JS, Prichard L, Schaper F, Schmitz J, Stephenson-Famy A, Rosenfeld ME, et al. Expression of suppressors of cytokine signaling during liver regeneration. J Clin Invest 2001;107:1285–92. [47] Reyes-Gordillo K, Segovia J, Shibayama M, Vergara P, Moreno MG, Muriel P. Curcumin protects against acute liver damage in the rat by inhibiting NF-κB, proinflammatory cytokines production and oxidative stress. Biochim Biophys Acta 2007;1770:989–96.

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