Extracellular Vesicles Derived from Human ...

ORIGINAL RESEARCH ARTICLE Extracellular Vesicles Derived from Human Embryonic Stem Cell-MSCs Ameliorate Cirrhosis in Thioacetamide-Induced Chronic Liver Injury† Running title: Improvement of Cirrhosis in TAA-Induced Chronic Liver Injury by ES-MSC EV Soura Mardpour1,2, Seyedeh-Nafiseh Hassani2, Saeid Mardpour3, Forough Sayahpour2, Massoud Vosough2, Jafar Ai1, Nasser Aghdami2, Amir Ali Hamidieh4*, Hossein Baharvand2,5* 1. Tissue engineering and Applied Cell Sciences Department, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran. 2. Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran. 3. Department of Radiology, Kasra Hospital, Karaj, Iran. 4. Pediatric Stem Cell Transplant Department, Children's Medical Center, Tehran University of Medical Sciences, Tehran, Iran. 5. Department of Developmental Biology, University of Science and Culture, Tehran, Iran *Corresponding Addresses: Hossein Baharvand, PhD, Email: [email protected] And Amir Ali Hamidieh, MD, E-mail: [email protected]

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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/jcp.26413] Additional Supporting Information may be found in the online version of this article. Received 6 November 2017; Accepted 19 December 2017 Journal of Cellular Physiology This article is protected by copyright. All rights reserved DOI 10.1002/jcp.26413

This article is protected by copyright. All rights reserved

Abstract Various somatic tissue-derived mesenchymal stromal cells (MSCs) have been considered as an attractive therapeutic tool for treatment of liver diseases in which the secretion of soluble factors or extracellular vesicles (EVs) is the most probable mechanism. The experimental application of human embryonic stem cells-derived MSC (ES-MSC) increased rapidly and showed promising results, in vitro and in vivo. However, possible therapeutic effects of human ES-MSC and their EVs on Thioacetamide (TAA)-induced chronic liver injury have not been evaluated yet. Our data indicated that human ES-MSC can significantly suppress the proliferation of peripheral blood mononuclear cells compared to bone marrow (BM)-MSC and adipose (AD)-MSC. Moreover, ES-MSC increased the secretion of anti-inflammatory cytokines (i.e. TGF-β and IL-10) and decreased IFN-γ, compared to other MSCs. ES-MSC EVs demonstrated immunomodulatory activities comparable to parental cells and ameliorated cirrhosis in TAA-induced chronic rat liver injury, i.e., reduction in fibrosis and collagen density, necrosis, caspase density, portal vein diameter and transaminitis. The gene expression analyses also showed upregulation in collagenases (MMP9 and MMP13), antiapoptotic gene (BCL-2) and anti-inflammatory cytokines (TGF-β1 and IL-10) and downregulation of major contributors to fibrosis (Col1α, αSMA and TIMP1), pro-apoptotic gene (BAX) and pro-inflammatory cytokines (TNFα and IL-2) following treatment with ES-MSC and ESMSC-EV. These results demonstrated that human ES-MSC and ES-MSC EV as an off-the-shelf product, that needs further assessment to be suggested as an allogeneic product for therapeutic applications for liver fibrosis. This article is protected by copyright. All rights reserved

Keywords: Cirrhosis; Extracellular vesicles; Human Embryonic stem cells; Mesenchymal stromal cells; Liver


Introduction Chronic liver disease (CLD) which is often characterized by established cirrhosis accompanied by chronic inflammation, progressive destruction of liver structure, portal hypertension and liver dysfunction, is considered a life-threatening state and increases the risk of hepatocellular carcinoma (HCC) (Forbes and Newsome, 2016; Huber et al., 2015). Currently, orthotropic liver transplantation is the only effective treatment available for patients with end-stage cirrhosis. Post-operation complications and limited number of liver donors have forced researchers to find alternative therapeutic approaches. Effectiveness of mesenchymal stromal cells (MSCs) against liver diseases has been demonstrated in experimental models (Meier et al., 2015; van Poll et al., 2008) and later in clinical trials (Mohamadnejad et al., 2007; Nicolas et al., 2016; Pan et al., 2014; Peng et al., 2011; Shi et al., 2012; Squillaro et al., 2016; Suk et al., 2016; Vosough et al., 2016). MSCs promote hepatic regeneration through hepato-protective cytokines and reduce liver inflammation with the help of anti-inflammatory and immunomodulatory properties of these cytokines (Forbes and Newsome, 2016; Than et al., 2016). Despite the advantages of somatic tissue-derived MSCs, they show some drawbacks such as the need for a consistent source of cells and high cost of handling and maintenance. An alternative source of MSCs could be human pluripotent stem cells including embryonic stem cell (ES-MSC) or induced pluripotent stem cell (iPS-MSC) that can overcome some challenges facing clinical application of somatic tissue-derived MSCs. However, cell rejection, ectopic tissue formation and infusion toxicity due to lodging of injected cells in the pulmonary capillaries are still problematic.


Recent evidence suggest that bilayer membranous extracellular vesicles (EVs) (i.e., exosomes (40-100 nm in diameter) and microvesicles (MVs; 0.1-1 mm in diameter) (Bruno et al., 2015; Rani et al., 2015) that are secreted by MSCs, might constitute a convincing alternative cell-free therapy because of their advantages over the mentioned MSCs (Lou et al., 2017). EVs contain mRNAs, microRNAs and proteins. Previous studies demonstrated that MSC-EVs could improve acute liver injuries and increase survival rate after lethal hepatic failure (Haga et al., 2017b), hepatic oxidant injury (Yan et al., 2017), hepatic ischemia-reperfusion injury (Haga et al., 2017a; Nong et al., 2016), fulminant hepatic failure (Chen et al., 2017a). Accelerated liver regeneration (Tan et al., 2014) following MSC-EVs injection suggests that the content of these cells might be critical for reversing liver injury. Using a model of CCl4-induced acute fibrotic liver injury, it was shown that direct injection of human umbilical cord-derived MSC-EV alleviated hepatic inflammation and collagen deposition (Li et al., 2013b). However, no study has reported the application of human ES-MSC EV and their parental cells (ES-MSC) in a thioacetamide (TAA)-induced chronic liver injury model. We evaluated the immunomodulatory effect of BM-, AD- and ES-MSC and investigated the therapeutic potential of human ES-MSC and ES-MSC EVs. The immunomodualatory effects on peripheral blood mononuclear cells (PBMCs) were assessed in vitro and in a rat model of TAAinduced chronic liver fibrosis. The results demonstrated the therapeutic effects of human ESMSC and ES-MSC EVs as an off-the-shelf product and an allogeneic cell source for treatment of chronic liver fibrosis.


Materials and methods Generation and characterization of ES-MSCs Human embryonic stem cell line, Royan H6 (Baharvand et al., 2006) (Royan Stem Cell Bank, Tehran, Iran) was cultured on matrigel-coated plates in Dulbecco’s modified Eagle’s medium/F12 (Life Technologies) supplemented with 20% knockout serum replacement (Life Tehnologies), 2 mM GlutaMAX, 0.1 mM non-essential amino acids (NEAAs), 1% insulintransferrin-selenium (ITS) (all from Gibco, USA), 0.1 mM β-mercaptoethanol (Sigma-Aldrich, USA) and 100 ng/ml basic fibroblast growth factor (bFGF, Royan Biotech, Tehran, Iran) as described before. Every other day, the medium was changed and every five days the colonies were splitted using collagenase IV (Sigma-Aldrich, USA).

In order to induce ESCs

differentiation into MSCs, spontaneous differentiation was performed. Briefly, ESC colonies were dissociated using 0.05% trypsin/EDTA (Gibco, USA) and plated on 6-cm2 non-adherent culture petri dish at 2×105 cells/cm2 in ESC medium supplemented with 10 µM ROCK inhibitor Y-27632 (Calbiochem, USA) in the absence of bFGF. The medium was refreshed every other day to generate embryoid bodies (EBs) after 7 days. Then, generated EBs were plated on gelatincoated plates in Alpha Modified Eagle’s Medium (α-MEM, Life technologies) supplemented with 10% fetal bovine serum (FBS, Hyclone, USA) and 2 mM L-glutamine for 7-10 days. The outgrowing differentiated cells were collected and subsequently re-plated and cultured for additional days. Then, resulted ES-MSCs of passage 0 were passaged at 80% confluency until 16 passages. Expanded ES-MSCs of passage 3 were used for in-vitro and in-vivo experiments. The conditioned medium of ES-MSCs of passage three to seven was collected for EV purification. For growth rate, 1×104 cells/cm2 were cultured in T25 cm2 tissue culture flasks (TPP, Germany). Doubling time was calculated according to the following formula:


Doubling Time = duration∗ log (2)/ log (Final Concentration) – log (Initial Concentration).

The human BM- and AD-MSCs were provided by Royan Stem Cell Bank and cultured in Alpha Modified Eagle’s Medium (α-MEM, Life technologies) supplemented with 10% fetal bovine serum (FBS, Hyclone, USA) and 2 mM GlutaMAX at the same density.

Multilineage differentiation Osteogenic and adipogenic differentiation capacity of ES-MSC were evaluated 14 and 21 days post-incubation in osteogenic and adipogenic induction medium, as described before (Lotfinia et al., 2016).

Mixed lymphocyte reaction (MLR) assay To evaluate immunomodulatory effect of ES-MSCs, 1×105 cells were exposed to mitomycin-C (10 µg/ml) for 2 hours. Then, mitomycin-C-treated ES-MSCs were seeded on a 96 well plate for 24 hours. In addition, we isolated PBMCs from two HLA-mismatched donors by Ficoll-PaqueTM density gradient protocol. As we described before (Pourgholaminejad et al., 2016), the proliferation of allogenic stimulator PBMCs (S), was suppressed with mitomycin-C (25 µg/ml) following 45 minute treatment. Responder PBMCs (R) were incubated with 25 µM Carboxyfluorescein succinimidyl ester (CFSE) (Molecular probe, Invitrogen) for fluorescence labeling. For MLR assay, three experimental groups were considered (i.e. R, R+S, R+S+ESMSCs). For group R, only 1×105 responder PBMCs were cultured as control negative. In group R+S, both stimulator and responder cells were co-cultured together and considered as positive control. In R+S+ES-MSCs group, we co-cultured R+S in the presence of pre-seeded ES-MSCs in which the proliferation of responder PBMCs was detected based on the dilution of CFSE,


using flow-cytometry analysis (BD FACS caliberTM cytometer and FlowJo 7-6-1 software). In order to show the EVs-induced immunosuppression, we performed another experiment in which 10 µg of ES-MSC EVs were used instead of cells. Finally, three groups of R, R+S and R+S+ESMSC EVs were compared to each other regarding the percentage of the proliferated PBMCs.

Purification and characterization of EVs To isolate EVs, passage 3-7 of ES-MSCs were cultured in T150 culture flasks up to 70% cell confluency. Then, FBS-enriched medium was replaced with MSC basal medium supplemented with EV-free FBS plus 2mM GlutaMAX. Forty-eight hours post incubation with EV-free medium, the conditioned medium (CM) was collected and stored at -80˚C. To purify EVs, CM were submitted to two light spin centrifugation (300g for 10 min and 2500g for 25 min at 4˚C) to remove debris and dead cell bodies. Then, the remaining supernatant was centrifuged at 20000g for 25 min to isolate micro-sized vesicles. After that, the supernatant was subjected to ultracentrifugation at 100000 g for 2 hours at 4˚C. At this point, the supernatant was discarded and the EV pellet was washed with PBS using ultracentrifugation for additional 2 hours similar to the previous step. Finally, the supernatant was removed and the pellet was re-suspended in PBS and stored at -80˚C. We used fixed angle Type 45Ti rotor and Optima™ L-100XP ultracentrifuge instrument. The protein concentration of EVs was measured using BSA protein assay kit (Pierce, ThermoFisher). To determine the size and relative intensity of ES-MSC EVs, we used dynamic light scattering (DLS) equipment (Malvern, UK). The spheroid morphology of vesicles was determined using scanning electron microscopy. Moreover, the expression of general EV markers including CD81, CD63 and TSG101 were evaluated using western blot standard protocol described by Thery et al (Thery et al., 2006). In order to do a qualitative


assessment of targeted delivery and engraftment of EV to the liver, the EV pellet was suspended in 1 ml diluent C in a vial and 4 µl PkH-26 red florescence linker membrane lipophilic dye was added to 1 ml diluent C in another vial and mixed well to disperse. After 5 minutes of incubation, staining was stopped using 1%BSA and it was subjected to additional ultracentrifugation to pellet EVs. Then, PKH-labeled EVs were used for in-vivo engraftment and also for in-vitro primary hepatocyte uptake. They were observed using fluorescence microscope (IX71; Olympus) and KODAK in-vivo F series imaging system.

Histopathological analysis Histopathology analyses performed by two blinded pathologists on four slides for each group. The liver tissue was fixed using 10% formalin, processed, and paraffin-embedded. Then, 6-µm thickness tissues were prepared using microtome and subsequently stained with hematoxylin and eosin (H&E) and masson-trichrome (MT). Histopathology data according to histological grading and staging (Ishak’s score) were classified to piecemeal necrosis (grade 0-4), confluent necrosis (grade 0-6), focal lytic necrosis (grade 0-4), portal inflammation (grade 0-4) and fibrosis (grade 0-6).

Animal models Male wistar rats (150 to 200 g body weight) were kept in single cages under 12 hour light/12 hour dark cycles and controlled humidity and temperature condition in Royan animal facility. To induce cirrhosis, all animals received intraperitoneal injection of 200 mg/kg thioacetamide (TAA) twice weekly for 16 weeks. Ultrasound imaging was performed for confirmation of cirrhosis after 16 weeks. Then, cirrhotic animals were assigned to four groups including ES-


MSC (n=4), ES-MSC EVs (generated from the same lot of the cells; n=4), fluorescence labeled ES-MSC EVs (n=1), vehicle (received PBS; n=4) against normal (n=3). Next, 4×106 cells and 350 µg EVs were dissolved in 400 µl injection water and infused intra-splenicly under the guidance of ultrasound. To evaluate targeted delivery of vesicles, fluorescence-labeled ES-MSC EVs were injected and animals were subjected to KODAK in-vivo F series for live imaging 24 hour-post administration. Other animals were followed up for four weeks. After sonography imaging at week 20, animals were euthanized, the excised liver tissues were deep freezed in liquid nitrogen and animal sera were collected for additional assays. All animal studies and procedures were already approved by the Royan Institutional Review Board

and

Institutional

Ethics

Committee

of

Royan

Institute

(No:

IR.ACECR.ROYAN.REC.1394.11).

Statistical analysis All experiments were conducted in at least three independent repeats. All data were showed as mean ± SD or mean ± SEM and one way analysis of variance (ANOVA) was used to determine differences between groups with Tukey post-hoc test and the viability with student’s t test. Pvalues < 0.05 were considered significant.

Results Generation and characterization of ES-MSCs In order to produce ES-MSCs, human ES cells were cultured in ES medium in the absence of bFGF to form EBs. Then, EBs were plated in gelatin-coated plates and cultured in MSC medium. Spontaneous differentiating of EBs resulted in outgrowth of ES-MSCs. They were further


passaged in order to acquire a homogenous population that had spindle-shaped morphology (Fig. 1A). The resulting cells could be passaged 16 times. The procedure of MSCs isolation from ESCs is reproducible. The morphology and yield in independent experiments were similar. These cell lines were then passaged 14 times without noticeable differences in doubling time (Fig. 1B) and fold changes (Fig. 1C). The growth rate of the cells started to decrease at passage 14 in both lines (Figs. 1B and 1C). Then, we characterized ES-MSC line 1. ES-MSCs at different passages were evaluated to characterize their surface antigen markers (Fig. 1D and Supplementary Fig. 1A). ES-MSCs expressed CD90, CD105 (endoglin, SH2), CD106, CD73 (SH3), and CD44. These cells were negative for CD34, CD45, and CD11b. This consistent antigen expression profile maintained intact in all consecutive passages. ESC markers, SSEA-4, OCT4 and Nanog did not express on ES-MSCs (Fig. 1D and Supplementary Fig. 1A, B). To confirm the multipotency of ES-MSCs, we used osteogenic and adipogenic media to differentiate them. After 3 weeks, the majority of the cells were differentiated into osteoblasts (Fig. 1E). Adipogenic differentiation demonstrated the accumulation of small cytoplasmic vesicles that are lightly stained by oil red O (Fig. 1F). For further characterization, the expression profile of differentiated cells were assessed using qRT-PCR. The results showed upregulation of transcription factors involved in osteogenesis and adipogenesis [bone-specific alkaline phosphatase (ALP), Collagen 1α (COL1α), Osteocalcin (OCN) and RUNX2) depicted in Fig. 1G and lipoprotein lipase (LPL), PPARγ and Adiponectin depicted in Fig 1H].


ES-MSCs were also not able to stimulate PBMC response in co-culture condition (Supplementary Fig. 3A). The immunophenotyping data of ES-MSCs and their similar differentiation capacity revealed that these cells presented the characteristics of MSCs.

ES-MSCs showed better immunomodulatory activity compared to BM- and AD-MSCs MSCs should be able to modulate the activity of immune cells directly by regulating inflammatory cytokine secretion (Uccelli et al., 2008). To assess this, we analyzed the proliferation of stimulated PBMCs co-cultured with ES-MSCs as well as BM or AD-derived MSCs and detected CFSE dilution using flow cytometry following MLR assay (Fig. 2A and Supplementary Fig. 2A). CFSE labeled-PBMCs of the first donor were stimulated to proliferate using a second donor PBMC population according to allogenic mixed lymphocyte reaction (MLR) assay. After IL2 stimulation, in the absence of MSCs, PBMCs as responder cells were highly proliferated. However, co-culturing with MSCs from different sources resulted in inhibition of proliferation of stimulated PBMCs (R+S) (a: p