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Title: Differential Reduction of Reactive Oxygen Species by Human Tissue-Specific
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Mesenchymal Stem Cells from Different Donors Under Oxidative Stress
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Authors: Swati Paliwal1, Anupama Kakkar1, Rinkey Sharma1, Balram Airan2 and Sujata
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Mohanty1#
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Affiliation: 1Stem Cell Facility, All India Institute of Medical Sciences, New Delhi, India.
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Delhi, India.
Department of Cardio Thoracic Vascular Surgery, All India Institute of Medical Sciences, New
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Running Title: Differential Reduction of ROS by Tissue-Specific MSCs
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#Corresponding
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Dr. Sujata Mohanty
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Additional Professor
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Stem Cell Facility
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DBT Centre of Excellence for Stem Cell Research
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All India Institute of Medical Sciences
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New Delhi: 110 029, India.
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Email: [email protected], [email protected]
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Phone: +91-11-2659 3085, Fax: +91-11-2658 8663/641
Author
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ABSTRACT:
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Clinical trials using human Mesenchymal Stem Cells (MSCs) have shown promising results in
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the treatment of various diseases. Different tissue sources such as bone marrow (BM-MSCs),
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adipose tissue (AD-MSCs), dental pulp (DP-MSCs), umbilical cord (UC-MSCs), are being
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routinely used in regenerative medicine. MSCs are known to reduce increased oxidative stress
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levels in pathophysiological conditions. Differences in the ability of MSCs from different donors
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and tissues to ameliorate oxidative damage have not been reported yet. In this study, for the first
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time, we investigated the differences in the reactive oxygen species (ROS) reduction abilities of
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tissue-specific MSCs to mitigate cellular damage in oxidative stress. Hepatic Stellate cells (LX-2)
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and cardiomyocytes were treated with Antimycin A (AMA) to induce oxidative stress and tissue
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specific MSCs were co-cultured to study the reduction in ROS levels. We found that both donor’s
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age and source of tissue affected the ability of MSCs to reduce increased ROS levels in damaged
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cells. In addition, the abilities of same MSCs differed in LX-2 and cardiomyocytes in terms of
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magnitude of reduction of ROS, suggesting that the type of recipient cells should be kept in
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consideration when using MSCs in regenerative medicine for treatment purposes.
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Key Words: Tissue-Specific Mesenchymal Stem Cells, Oxidative Stress, And Reactive Oxygen
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Species
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INTRODUCTION:
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Mesenchymal stem cells have become a popular choice for clinical trials owing to their immuno-
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modulatory activities, low or no risk of rejection, and their easy availability. They possess the
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self-renewable capacity and ability to differentiate into multiple lineages. MSCs can be derived
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from various tissue sources such as bone marrow (BM), adipose (AD), dental pulp (DP) and
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umbilical cord (UC). Tissue-specific MSCs vary in their proliferative capacity, population
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doubling time (PDT) and differentiation potential (Kern et al. 2006, Chen, Mou et al. 2015, Li,
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Wu et al. 2015). Several mechanisms such as paracrine secretion, mitochondrial transfer,
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exosome secretion, cell fusion and trans-differentiation contribute towards regeneration of
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damaged cells (Ahmad et al. 2014 , Koyanagi et al. 2005, Caplan and Dennis 2006, Bruno et al.
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2009, Islam et al. 2012). Recent studies have contributed to the dramatic shift in paradigm
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suggesting that MSCs regulate repair mechanism primarily by modulating inflammation and
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immune response by paracrine secretion and via neutralization of increased ROS levels in cells
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under oxidative stress (Park et al. 2009, Ranganath et al. 2012, Maumus et al. 2013, Raposo and
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Stoorvogel 2013, Ronne Wee Yeh et al. 2013, Liang et al. 2014). Studies have shown that tissue-
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specific MSCs differ in their immuno-modulatory activities, variation in secretion of cytokines,
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growth factors and other components of their secretome (Jin et al. 2013, Collins et al. 2014, Pires,
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Mendes-Pinheiro et al. 2016). Differences in these properties impact their regenerative potential
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in clinical applications has been observed (Jin et al. 2013, Collins et al. 2014). But, the
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differences in differential regulation of oxidative stress by tissue-specific MSCs have not been
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studied yet.
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Few reports have suggested that various factors such as tissue-source, donor’s age, passage
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number, conditioned media and health of donor affect the properties of tissue-specific MSCs
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(Collins et al. 2014, Nakamura et al. 2015, Khatri et al. 2016). But, the effect of these factors on
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MSCs in a co-culture system with different recipient cells and in variety of pathological
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conditions has not been explored well. Generation of free radicals causes oxidative stress that
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leads to diverse pathophysiological conditions (Griendling and FitzGerald 2003). Many diseases
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such as cardiovascular diseases, neurodegeneration, renal disorder and liver fibrosis are mediated
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through the excess accumulation of ROS (Poli 2000, Griendling and FitzGerald 2003). Treatment
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of cells with AMA is known to induce oxidative stress by the generation of superoxide anion,
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decreased respiration and cellular damage (King and Radicchi-Mastroianni 2002, Dutta, Xu et al.
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2013). Thus, AMA induced oxidative stress in cells such as cardiomyocytes, hepatocytes and
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epithelial cells were considered as a model to understand diseases prognosis and treatment
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modalities (Ahmad, Mukherjee et al. 2014 , Dutta et al. 2013, Kawano et al. 2014). The
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antioxidant effect of MSCs to mitigate oxidative damage has been reported in many studies
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(Ahmad, Mukherjee et al. , Caicedo, Fritz et al. 2015, Phinney, Di Giuseppe et al. 2015). MSCs
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reduce increased ROS levels by regulating several factors participating in stress pathways,
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thereby, promoting healing and rejuvenation of injured cells (Cho et al. 2012, Liu et al. 2012,
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Ohkouchi et al. 2012).
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In this study, we have investigated the differences in tissue-specific MSCs to alleviate oxidative
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stress damage in terms of reduction ROS levels. Considering that oxidative stress is a mediator of
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liver fibrosis and cardiovascular diseases, hepatic stellate cells (LX-2) and cardiomyocytes were
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selected as representatives and were treated with AMA to induce oxidative stress. Post AMA
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treatment, these cells were co-cultured with tissue specific MSCs (BM-MSCs, AD-MSCs and
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DP-MSCs) separately to study the attenuation of elevated ROS levels in cells under oxidative
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stress.
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2. METHODS
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2.1 Revival and Characterization of Cryopreserved human MSCs
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This study was ethically approved by theInstitutional Committee for Stem Cell Research (IC-
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SCR), All India Institute of Medical Sciences (AIIMS), New Delhi. MSCs were isolated from
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respective tissue samples and cryopreserved after taking prior informed consent from the the
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MSCs donors. The details of cryopreserved (5 years) human BM-MSCs, AD-MSCs and DP-
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MSCsare provided in Supplementary Table 1. All the MSCs used in the experiments were
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revived in LG-DMEM (Gibco, USA) media containing 10% Fetal Bovine Serum (FBS; Hyclone)
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supplemented with 1% Glutamax & penstrep (Invitrogen, USA), incubated at 37°C with 5% CO2.
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In-vitro culture expansion and characterization of MSCs and viability test were done as per
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previously described lab protocol (Mohanty, Bose et al. 2013, Nandy SB 2014). Surface marker
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profiling of CD73 PE, CD90, PECy5, HLA Class I APC, HLA Class II FITC, CD 34 PE (Becton
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Dickinson, USA) & CD105 APC (eBioscience, USA) and tri-lineage differentiation of MSCs to
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osteocyte, chondrocyte and adipocytes was performed ( Mohanty, Bose et al. 2013). Controls
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were made by corresponding isotypes: IgG1 coupled with PE, PECy5, APC and FITC. The cells
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were acquired on BD LSR II flow cytometer (Becton Dickinson, USA) with at least 10,000
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events for each sample and analyzed with Becton Dickinson FACS Diva (ver 6.1.2) for surface
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marker characterization (Nandy et al. 2014, Kakkar et al. 2015). Cells at passage 3 were used for
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all further experiments.
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2.2 Cell Cultures
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Human hepatic stellate cells LX-2 cell line were maintained in DMEM media with 2% FBS and
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supplemented with penicillin (200 μg/ml), streptomycin (200 U/ml) and 1X antimycotic solution,
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Amphotericin B (Gibco, USA). Human Cardiac biopsy obtained from the Department of Cardio
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Thoracic Vascular Surgery (AIIMS, New Delhi) were cultured for cardiomyocytes and
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cryopreserved. The cryopreserved cultures were revived as per the standard lab protocol and
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maintained in DMEM/Ham F12 in ratio 3:1 with 10% FBS. Characterization of cardiomyocytes
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was performed using flow cytometry and Immunofluorescence for Myosin light chain-2v (Mlc-
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2v) and Cardiac troponin I (cTnI) as per our established protocol (Kakkar et al., 2015).
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2.3 Oxidative Stress Induction
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AMA (Sigma, USA) at 100nM was added to culture media of LX-2 and cardiomyocytes (50,000
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cells/well in 12 well culture plate) and incubated at 37ºC and 5% CO2for 18 hours to induce
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oxidative stress. After 18 hours, cells were washed with 1xPBS (pH 7.4) before adding media and
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MSCs for co-culture experiments.
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2.4 Co-culture
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Three different tissue-specific MSCs (BM-MSCs, AD-MSCs and DP-MSCs) were trypsinized
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and co-cultured with AMA treated LX-2 or cardiomyocytes, at 1:1 ratio and added equal amounts
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of respective media and incubated at 37ºC with 5% CO2 for 24 hours.
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2.5 Quantification of ROS
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ROS was measured using MitoSOX red (Life technologies, USA) at a concentration of 4 µM for
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20 minutes in respective media and incubated at 37°C with 5% CO2. The quantification of ROS
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was done using flow cytometer BD LSR II flow cytometer (Becton Dickinson, USA) with at least
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20,000 events for each sample and analyzed with Becton Dickinson FACS Diva (ver 6.1.2).
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Readings (in duplicates) for mean fluorescence intensity (MFI) in PE region was recorded in
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arbitrary units (AU). Control comprised of MSCs + LX-2 (or cardiomyocytes) unstained and the
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MFI which was subtracted from co-culture with MSC + LX-2 (or cardiomyocytes) treated with
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AMA.
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2.6 ATP Detection
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ATP detection was performed using ATP Assay Kit as per manufacturer’s instructions (Abcam,
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USA). Cells were grown in duplicates in 12-well plate for co-culture. Each well contained total
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5x105cells that were used for ATP assay. After 24 hours of co-cultures, cells were lysed in the
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ATP assay buffer and centrifuged at 15,000 g for 2 min. The supernatant was added to a 96-well
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plate and followed by the addition of 50 μl of the reaction mix to each well and detected using
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colorimetric detection by spectrophotometer (BioTek, USA) at 570nm. Standard curve was
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prepared using ATP standard provided in the kit. Relative amount of ATP in samples was
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calculated using standard curve.
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2.7 Fluorescence Imaging
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Tissue-specific
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(Excitation/Emission: 419/560nm) from Thermo Fisher, Scientific and LX-2 cells were stained
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with Tetramethylrhodamine ethyl ester (TMRE) from Thermo Fisher, Scientific) using EVOS®
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FLoid® Cell Imaging Station (Life Technologies, Carlsbad, CA, USA).
MSCs
were
stained
by live
imaging
by MitoTracker
Green
FM
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2.8 Statistical Analysis
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Statistical analyses were performed using R 2.13.0 and Minitab software (R Development Core
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Team 2010). One-Way and Two-Way ANOVA analysis was done to determine the effect of
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tissue- specific MSCs and donors MSCs (Significant values, P value <0.05).
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3. RESULTS:
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3.1 Differential Reduction of ROS by Tissue-Specific MSCs in LX-2 Cells under Oxidative
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Stress
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Cryopreserved MSCs were successfully revived and characterized based on the presence of all
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surface markers including CD105, CD73 PE, CD90 PECy5, HLA Class I APC and absence of
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HLA Class II FITC, CD 34/45 PE/FITC (Becton Dickinson, USA) (figure 1A).Viability and
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trilineage differentiation potential of MSCs were also confirmed (figure 1B). Florescence images
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of tissue-specific MSCs labeled with Mitotracker Green FM and LX-2 cells with TMRE
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demonstrated healthy cells (figure 2A). The shift in ROS levels of LX-2 before and after
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treatment of AMA and post co-culture with tissue-specific MSCs in PE region by flow cytometry
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data depicted lowering of ROS levels by MSCs (figure 2B). The base level ROS of LX2
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increased significantly from average MFI 2592AU to 4092 AU after oxidative stress induced by
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treatment of AMA, P<0.05 (figure 2C). Overall, average of all tissue-specific MSCs were able to
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reduce ROS by 72.88% in LX-2 cells. Differential ROS reduction abilities of ten donor tissue-
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specific MSCs, numbered from 1 through 10, was observed in their co-culture with LX-2 (AMA)
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(figure 2C). Average percentage reduction by BM-MSCs (n=3), was found to be 64.09%, AD-
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MSCs (n=4) was 68.61% and by DP-MSCs was 87.4% (figure 2C). Lower values of ROS in the
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bar graphs demonstrate higher reduction ability in co-cultures with LX-2 (AMA). The reduction
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in ROS by all three tissue-specific MSCs was found to be significantly different and the reduction
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abilities were noted as DP-MSCs> AD-MSCs>BM-MSCs.
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3.2 Rescue Abilities of MSCs are affected both by Donor’s Age and Tissue Source
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Donor MSCs were divided into two groups, younger age group (10-30 years, n=6) and older age
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groups (31-60 years, n=4), details are given in Supplementary Table S1. It was also found that
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ROS values of young MSCs donors were lower than the old donors indicating their better ability
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to reduce ROS (figure 3A). The average amount of ROS reduction amount in young donor group
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was found to be higher in DP-MSCs and in the following order: DP-MSCs>AD-MSCSs>BM-
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MSCs. Older age BM-MSCs showed the lowest reduction capacity. Also, older AD-MSCs were
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found to reduce more ROS than older BM-MSCs (figure 3A). One-Way ANOVA clearly
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demonstrated that donor source significantly impacts ROS reduction capacity, P<0.01.
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Interestingly, Two-Way ANOVA analysis demonstrated the equal contribution of both donor and
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tissue source in reducing ROS levels, P <0.05 (figure 3B). Interaction plots showed the effect of
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donor type and each tissue-specific MSC and their combined contributions in affecting ROS
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levels (figure 3B).
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3.3 Rescue Effect of MSCs on Cardiomyocytes
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Cardiomyocytes were characterized for Mlc-2v (78.5%) and cTnI (77.5%) using flow cytometry
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and fluorescent microscope imaging with cardiomyocytes specific-markers (figure 4). We
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observed that all three MSCs type were able to differentially reduce ROS levels, also in
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cardiomyocytes under oxidative stress (figure 5A). The shift in the mean fluorescence intensity
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under oxidative stress and post-culture with tissue-specific MSCs is shown by flow cytometry
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data (figure 5A). All the tissue-specific MSCs were able to reduce elevated levels of ROS, and
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total average reduction of ROS in cardiomyocytes was noted as 61.29%. It was observed that
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overall co-cultures of stressed cardiomyocytes with DPMSCs (71.27%), demonstrated lowest
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absolute ROS levels followed by AD-MSCs (67.3%) and BM-MSCs (37.29%), (Fig. 5B).
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ANOVA analysis revealed significant differences in the reduction capacities of tissue-specific
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MSCs, P value<0.05. Thus, although average ROS reduction abilities of DP-MSCs>AD-
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MSCs>BM-MSCs remained similar but, if considered donor-wise comparisons of MSC donors (8
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of the 10 donors depicted by numbers) in both LX-2 and cardiomyocytes a different pattern in
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their reduction abilities was observed along with noted changes in magnitude difference in ROS
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reduction abilities, figure 6.
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3.4 No Correlation between Reductions in ROS and ATP Production
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We expected that lowering in ROS would be accompanied by a subsequent increase in ATP
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levels and tested it in two young donors chosen from each tissue-specific MSCs (total n=6).
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Although, we found that when MSCs were co-cultured with cardiomyocytes (AMA treated),
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there was subsequent increase in ATP along with the decrease in ROS (figure 7), but did not find
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any significant correlation between reduction in ROS and increase in ATP values.
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4. DISCUSSION
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Effective clinical outcomes demand the use of optimal source of MSCs that can lead to consistent
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and better clinical outcomes. Although, MSCs are popularly being used for clinical trials but the
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availability of many options of tissue source makes it difficult to determine the optimal source
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that can be used in regenerative medicine. It has been challenging to identify suitable source of
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stem cells due to several confounding factors including the heterogeneous population of stem
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cells, different isolation procedures, variation in expansion media along with other confounding
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factors including donor’s age and tissue source of MSCs. Though, all the tissue-specific MSCs
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share few similar characteristic properties such as surface marker profiling but they vary in many
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other characteristics such as differential potential and secretion of immuno-modulatory factors
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(Jin et al. 2013). This further suggests that these MSCs also differ in their treatment efficacy on
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the basis of their origin and mechanism involved in rejuvenation of damaged cells (Collins et al.
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2014). One of the mechanisms through which MSCs alleviate cell damage under oxidative stress
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is by neutralization of enhanced ROS. Liver fibrosis and cardiovascular diseases are caused by
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oxidative damage and thus, reduction in ROS levels in stressed cells by tissue-specific MSCs
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may indicate their ability to ameliorate cellular damage. Several mechanisms of MSC action are
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known including anti-inflammatory activity, secretion of paracrine factors and reduction in
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enhanced ROS levels of damaged cells that contribute in repair and rejuvenation (Liu et al. 2012,
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Ohkouchi et al. 2012). In case of myocardial infarction, decrease in ROS levels is found to
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correlate with reduction in infarct size (McCully et al. 2009). Ahmad et al., have demonstrated
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that MSCs have ability to reduce ROS in case of acute lung injury along with increase in ATP
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production (Ahmad et al. 2014). Despite these evidences, the differential ability of tissue-specific
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MSCs to reduce excess ROS, in case of oxidative damage still remains unexplored. Till date, no
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study has compared the ROS reduction abilities of tissue-specific MSCs from multiple donors. In
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this study, we investigated differential ability of tissue-specific MSCs to reduce accumulation of
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ROS under oxidative stress condition in LX-2 and cardiomyocytes induced by AMA. Hepatic
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stellate cells are activated under oxidative stress that leads to liver fibrosis, thus AMA that
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inhibits complex III, leading to accumulation of free radicals was used to generate oxidative
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stress condition (Poli 2000, Proell et al. 2007, Bataller and Lemon 2012). In accordance, with
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findings of other research group showing reduction in ROS in other cells, we found that similar
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results when MSCs were co-culture with stressed cells (Ahmad et al. 2014). We also found
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differential lowering of ROS by tissue-specific MSCs obtained from multiple donors in co-
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cultures of LX-2 and cardiomyocytes under oxidative stress. Interestingly, we found that DP-
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MSCs were able to demonstrate more reduction in ROS as compared to BM-MSC and AD-MSCs
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(figure 2). It should be noted that we did not include any older age group for DP-MSCs due to
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lack of donor availability and this adds to limitation of our study along with non-inclusion of
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other favored tissue-specific MSCs such as UC-MSCs (Li et al. 2012, Jin et al. 2013). Our data
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demonstrates that younger donor were more efficient in reducing ROS than older MSCs donors
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(figure 2 A). This finding is in line with other studies that have shown that better proliferative and
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immuno-modulatory activities of younger MSCs as compared to MSCs from older age donors
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(Beane et al. 2014, Bruna et al. 2016, Khatri et al. 2016).
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In addition, two-way ANOVA reveals that both donors and tissue source significantly impacts
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ROS reduction capability of MSCs under oxidative stress (figure 2B). This suggests that both
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donor source and tissue are critical factors that affect use of MSCs for therapeutic application.
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Differential reduction in ROS was also observed in cardiomyocytes treated with AMA (figure 5).
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Interestingly the pattern of reduction of ROS was not same in co-cultures of stressed LX-2 and
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cardiomyocytes (figure 6). This suggests that the interaction between tissue-specific MSCs and
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the injured cell also matters and the origin or type of recipient should also be considered when
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using MSCs in regenerative medicine. Also, it indicates that a particular tissue specific MSCs
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may be able to ameliorate a disease condition better than other MSCs in specific injured tissues. It
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was found that all MSC have the capacity to reduce ROS under stress levels, however each has
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different capacity depending upon a particular given condition. It is important to note that, small
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sample size of can be considered as a limitation of this study and a larger data set would have
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provided robust statistical analysis. Nonetheless, these are first observation of this kind and can
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serve as proof of concept for future research along this direction.
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During oxidative stress induced by AMA, there is blockage of complex III of electron transport
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chain in mitochondria affecting the mitochondrial bioenergetics and ATP production (King and
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Radicchi-Mastroianni 2002). We expected that reduction would be accompanied by an increase in
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ATP indicating restoration of cellular health and mitochondrial bioenergetics. Although, we did
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find increase in ATP, after stressed cardiomyocytes were co-cultured with tissue-specific MSCs
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but the differences were not significant (figure 7). Also, no significant correlation was observed
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between reduction in ROS and increase in ATP. As DP-MSCs showed maximum reduction in
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ROS, we expected that they would lead to maximum ATP production. However, BM-MSCs were
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found to show most elevated levels of ATP as compared to AD-MSCs and DP-MSCs, which
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demonstrated comparable levels of ATP in co-culture (figure 7). It is plausible that other factors
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such as genetic components, mitochondrial health, basal ROS and ATP levels of MSCs,
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mitochondrial biogenesis, anti-oxidant mechanisms and paracrine factors might also be
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responsible for contributing ability to reduction in ROS levels.
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In conclusion, we found that tissue-specific MSCs differ in their capacity to reduce ROS in
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injured cell under oxidative stress. It was observed that younger donors were more efficient in
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reducing ROS as compared to MSCs obtained from older age donors. Thus, the source of donor
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MSCs and their age greatly impacts the ability to alleviate ROS in stress conditions. The rescue
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abilities of same MSCs also differed in magnitude and patterns of reduction for donor-specific
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MSCs in LX-2 and cardiomyocytes. This demonstrates that the interaction between donor MSCs
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and recipient cells should also be considered when using MSCs for clinical and therapeutic
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applications. This study suggests that these different parameters cannot be ignored when selecting
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the best-suited MSC candidate prior to using MSCs as off-the-shelf therapy in regenerative
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medicine. Further investigations, in this direction are warranted to increase our insight of these
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mechanisms and to identify an optimum source of MSCs in treatment of diseases manifested by
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oxidative stress.
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ACKNOWLEDGEMENT:
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We would like to thank Indian Council of Medical Research (ICMR) and Department of
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Biotechnology for providing funds and fellowship for conducting this work. We also thank Dr.
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M. Mani Sankar for providing inputs during scientific discussions.
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AUTHORS DISCLOSURE STATEMENT
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No competing financial interest exist
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