Tracking iron oxide labelled mesenchymal stem cells(MSCs) using magnetic resonance imaging (MRI) in a rat model of hepatic cirrhosis

Homing and tumor attenuation potential of BM-MSCs labelled with superparamagnetic iron-oxide nanoparticles (SPIONs) in a rat model of hepatic cirrhosis was evaluated. Rat BM-MSCs were derived, characterized and labelled with SPIONs (200 nm; 25 mg Fe/ml). Hepatic cirrhosis was induced in Wistar rats (n=30; 10/group) with carbon tetrachloride (CCl4; 0.3 mL/kg body weight) injected twice a week for 12 weeks. Group-I was administered vehicle (castor-oil) alone; Group-II received two doses of unlabelled BM-MSCs (3x106 cells) and Group-III received two doses of SPIONs labelled BM-MSCs (3x106 cells) via tail vein injection (0.5 ml) at weekly intervals. All animals were sacrificed after two weeks for histological, radiological and biochemical analysis. Derived BM-MSCs demonstrated MSCs related CD markers. Histology confirmed induction of hepatic cirrhosis with CCL4. Levels of alanine-aminotransferase, aspartate-aminotransferase,alkaline-phosphatase and gamma glutamyl-transferase returned to normal levels following treatment with BM-MSCs. Uptake and homing of SPIONs labelled BM-MSCs, and reduction in the size of cirrhotic nodules were confirmed using transmission electron microscopy and magnetic resonance imaging respectively. BM-MSCs reduced the pathological effects of CCL4 induced hepatic cirrhosis and labelling BMMSCs with SPIONs were non-toxic and enabled efficient tracking using non-invasive methods.

functions it is quite natural for the liver to be exposed to both microbial and toxic substances, which can lead to altered liver structure and function. Intake of alcohol, drugs, bacterial or viral infections can thus lead to liver injury and/or toxic hepatopathy [1]. When such injury becomes persistent or chronic it leads to liver fibrosis/cirrhosis and in later stages can also transform into benign or malignant cancer and therefore is a major clinical concern.
©Biomedical Informatics (2019) 2 Current therapies including both allopathic and natural remedies help restoration of the liver function by taking advantage of its inherent regeneration potential. However, the reversal of lost architecture is minimal to absent when the liver damage is in its advanced stages of fibrosis/cirrhosis leading on to liver cancer. Administration of either differentiated or undifferentiated stem cells to alleviate the underlying pathology is largely being explored [2]. Stem cells and/or their derivatives can be modified to act as pro-apoptotic, anti-angiogenic and anti-proliferative agents against different cancers. As such cellular fractions and/or drug loaded nanoparticles or scaffolds are used as therapeutic agents or drug carriers [3][4][5][6].
Direct local injection of stem cells or other therapeutic agents is always not possible. In many instances, the stem cells are administered via the common accessible routes of drug administration, either as intravenous, intraperitoneal or intraarticular injections. Stem cells have inherent migratory properties toward tumor cells [7]. In addition, the cross-talk between stromal derived factor-1 (SDF-1) and cytokines/chemokines help in migration of stem cells to the afflicted sites [8]. Although stem cells per se have inherent migratory properties toward tumor cells, in most cases it remains to be verified whether the stem cells reach their intended site of action. Tracking of these stem cells/cellular products using advanced labelling materials and techniques therefore becomes essential.
Of the various advanced imaging techniques with high safety profile, positron emission tomography (PET) and MRI are mostly preferred for in vivo imaging studies [9]. In comparison to PET, MRI is the most popular and is used in conjunction with magnetic labelling, and is a powerful technique for non-invasive tracking of cells in vivo [10,11]. Magnetic nanoparticles have been used for both diagnosis and treatment with promising results [12][13][14][15]. Most commonly used are the ferrite and iron oxide nanoparticles, which due to their superparamagnetic properties are utilized for diagnostic and therapeutic uses. Moreover, these nanoparticles have low toxicity, larger surface area to volume ratio, less sensitivity to oxidation and therefore superparamagnetic iron oxide nanoparticles (SPIONs) are promising candidates for use in biomedical applications including molecular imaging [16][17][18][19].
Although MSCs continue to be used for their therapeutic benefits against various diseases, it remains to be ascertained whether the delivered MSCs reach their intended site of action. As such in the present study we explored the therapeutic role of MSCs labelled with SPIONs in the amelioration of hepatic injury/cirrhosis in rats, through MRI tracking of cells labelled with iron oxide nanoparticles (SPIONs). The combination of cell labelling and MRI tracking in a time dependent manner with close follow up of the lesion size provide a novel, more objective assessment of the role of MSCs in disease control.

Methodology: Animals:
All animal experiments were conducted in accordance with the regulations of the international animal use and care committee and approved by the Institutional Research Ethics Committee, King Abdulaziz University (KAU), Jeddah, Saudi Arabia vide protocol number HA-02-J-008. Thirty female Wistar rats, 14 to 16 weeks that weighed between 300 to 400 grams were used in the study. All rats were housed in the animal holding unit of King Fahd Medical Research Center (KFMRC), KAU, under a 12 h light/dark cycle at a temperature of 25°C and relative humidity ranging from 60% to 70% throughout the experiment. The animals had free access to standard rat pellet and water ad libitum.

Culture of bone marrow mesenchymal stem cells (BM-MSCs):
BM-MSCs derived from the bone marrow washings of femur and tibia of 6-8 weeks old Wistar rats were used for iron oxide labelling and their tracking in vivo. The BM-MSCs were thawed and expanded in culture using Dulbecco's modified Eagle's media (DMEM) containing 10% fetal bovine serum (FBS), 2mM Lglutamine and antibiotics [penicillin (50IU/streptomycin (50µg/ml)].

CD marker analysis using fluorescent activated cell sorting:
Early passage (P2) of the derived BM-MSCs was analysed for CD marker expression. Briefly, the cells in culture were trypsinized, aliquoted into different tubes (1x10 5 cells/tube) in 3% FBS and labelled with the antibodies (10 µl each) namely, CD29, CD44, CD73, CD34 and CD45 (BD Biosciences, San Jose, CA). Following incubation with respective antibodies for 20 min at 4°C in the dark, the cells were centrifuged (1000 rpm x 5 min). The cells were then resuspended in 3% FBS before analysis using FACSAria III flow cytometer and FACSDIVA software (BD Biosciences, San Jose, CA).

Labelling of MSCs with iron oxide nanoparticles:
Iron oxide nano particles [Fluid MAG-nano particles (Chemicell, Berlin, Germany)] was used to label the BM-MSCs. Briefly, the BM-MSCs were counted and seeded at a density of 1x10 5 cells/well in a 24-well plate and incubated overnight in a CO2 incubator under standard culture conditions of 37°C and 5%CO2 in atmospheric air. Following fresh changes of medium, iron oxide nanoparticles (SPIONs) were added at a concentration of 25 µg Fe/ml. In addition, 0.75 mg/ml Poly-l-lysine was added and the cells cultured for 24 h. Fluid MAG-nano particles (Chemicell, Berlin, Germany) are ferrofluids, 200-nm-diameter particles consisting of an aqueous dispersion of magnetic iron oxides coated by hydrophilic polymers which protect them against aggregation by foreign ions [20]. Poly-l-lysine was utilized as a prospective vehicle for SPIONs transport into cells, and enable endocytosis of iron oxide nanoparticles by MSCs

Transmission Electron microscopy (TEM):
Briefly, both unlabelled and labelled BM-MSCs in culture were trypsinized, washed thrice with PBS, centrifuged, fixed in 2.5% glutaraldehyde and post-fixed in 2% osmium tetroxide. The samples were then dehydrated in graded series of ethanol and embedded in epoxy resin. Ultrathin (50 -70 nm) sections collected on metal mesh grid was stained and imaged using Philips CM100 TEM (Holland).

Induction of hepatic cirrhosis:
Hepatic cirrhosis was induced in all rats by subcutaneous injection of 0.3 mL/kg body weight of carbon tetrachloride (CCl4) in castor oil using previously established protocol [22]. CCl4 was administered thrice in the first week and thereafter twice weekly for 12 weeks. Following induction of hepatic injury, the rats were randomly divided into three different experimental groups of ten each. Group-I was administered vehicle alone and served as control; Group-II received two doses of unlabelled BM-MSCs (3x10 6 cells) and Group-III received two doses of SPIONs labelled BM-MSCs (3x10 6 cells) via tail vein injections (0.5 ml, PBS). The cells were injected once a week and each rat therefore received two cell administrations in total.

Liver histology:
Formalin fixed liver tissue from normal control, CCL4 and BM-MSCs treated groups were dehydrated in ethanol series, washed in xylene and embedded in paraffin wax. Tissue blocks were sectioned at 5-6 µM thickness, deparaffinized and stained with hematoxylin and eosin and analyzed under microscope (Olympus Instruments, Tokyo, Japan).

Quantitative Real Time-PCR (qRT-PCR):
Total RNA was extracted from the liver tissue of both control and MSCs treated rats using MagNA Pure compact RNA isolation kit (Roche, Penzberg, Germany) according to the manufacturer's protocol. First-strand cDNA synthesis was carried out using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Primers were designed using previously published primers, and the primer sequences are given in Table 1. The qRT-PCR analysis was performed using the ABI StepOnePlus TM Real-Time PCR System (Applied Biosystems, Foster City, CA) using TaqMan® Universal PCR Master Mixand relative quantification was performed using the comparative 2 -ΔΔ Ct method.

Genes
Primer sequences   (Figure 1a) and were plastic adherent. The BM-MSCs were positive for the CD makers CD29, CD44, CD73 and negative for CD34 and CD45 (Figure 1b-f), and could be differentiated into adipocytes, chondrocytes and osteocytes (Figure 1g-i). MSCs cultured in adipogenic media showed changes in morphology starting from 8-10 days and by the end of second and third week almost 90% of the cells demonstrated the characteristic vacuolations upon staining with Oil Red O (Figure 1g). Cells cultured in chondrogenic media lost their spindle shape and became polygonal and these cells demonstrated positive staining with Alcian blue which is indicative of collagen matrix deposition (Figure 1h). The MSCs cultured in osteogenic media showed dense deposits in culture which stained positive with Alizarin red indicative of calcium deposition (Figure 1i).

BM-MSCs labelling with SPIONs and TEM:
The SPIONs were readily taken up by the BM-MSCs and their cellular localizations were demonstrated by TEM. The SPIONs labelled BM-MSCs showed even distribution of the internalized nanoparticles in the cytoplasm which appeared as dark dense spherical bodies (Figure 2a, b).

BM-MSCs proliferation assay following labelling with SPIONs:
BM-MSCs cultured with SPIONs demonstrated normal cell proliferation like that of the control. No changes in morphology or any adverse effects leading to cell death were observed indicating that iron oxide cell labelling was effective without any compromise on cellular viability (Figure 2c).

Liver enzymes:
The levels of ALT and AST were increased in CCl4 treated group compared to the normal control. However, treatment with BM-MSCs led to decrease in both ALT and AST to near normal levels. The values of ALT were 82.0, 304.25, 89.25 mU/mL and AST were 41.25, 545.50, 182.0 mU/mL, for normal control, CCl4 treated group and BM-MSCs treated group respectively. There was a mean percentage increase in CCl4 treated group by 371.04% for ALT and 1322.42% for AST compared to normal control and these increases in values were statistically significant (Figure 3a). However, compared to the CCl4 treated group there was a mean percentage decrease by 70.67% for ALT and 66.64% for AST and these decreases were statistically significant (Figure 3a).  (Figure 3b). However, compared to the CCl4 treated group there was a mean percentage decrease by 38.03% for ALP and 75.96% for GGT and these changes were statistically significant (Figure 3b).

Liver injury/cirrhosis:
The liver samples from untreated controls showed normal histological pattern of liver lobules with radially arranged hepatocytes, the central portal vein and peripheral portal triad (Figure 4a). In contrast, the CCl4 treated group the liver lobules showed more trabecular pattern with loss of hepatocytes and more connective tissue infiltration (Figure 4b). However, the group treated with MSCs following CCl4 induced liver injury, showed reversal towards normal liver pattern (Figure 4c).

Figure 4:
Haematoxylin and eosin stained liver sections of normal (a), fibrotic liver following carbon tetrachloride (CCl4) injections (b) and regenerated liver following treatment with BM-MSCs (c). Dense areas of fibrosis (indicated by arrows) and disorganized liver pattern was observed following CCl4 induced liver injury (b), while normal hepatic architecture with radial arrangement of hepatocytes as that of normal liver was seen following liver regeneration with BM-MSCs treatment (c). Magnification 10x.

Magnetic Resonance imaging (MRI):
MRI done following administration of iron oxide labelled BM-MSCs demonstrated a sequential fall in liver intensity, compared to the unlabelled MSCs. In the unlabelled rats the signal intensities of the liver remained increased over the 6-day period of scanning (Figure 5a). In labelled rats, there was a sequential fall in the signal intensity of the liver (Figure 5a). Axial T2 Turbo RARE images done on rats in CCl4 treated groups demonstrated multiple dysplastic nodules in both lobes of a cirrhotic liver in the BM-MSCs preinjection scan (Day 0). Following administration of both unlabelled and labelled BM-MSCs, the size of these nodules decreased with time from 2.63 mm (day 0) to 1.39 mm (day 6) (Figure 5C). Sequential fall in the signal intensity of the liver nodules was noted in rats that received SPIONs labelled BM-MSCs, while the signal ©Biomedical Informatics (2019) intensity was increased in rats that received unlabelled BM-MSCs (Figure 5b).

Quantitative real-time PCR:
Real time gene expression analysis showed an increase in survivin and a decrease in β-catenin gene expression, while cyclin-D1 showed a biphasic change compared to the control. Survivin was increased by 1.5 fold in both Group-II and Group-III. Cyclin-D1 was increased by 1.3 fold and decreased by 0.63 fold in Group-II and Group-III respectively. β-catenin showed no changes in Group-II compared to control while it was decreased by 0.63 fold in Group-III. The observed differential expression of genes were however not statistically significant (Figure 6).

Discussion:
Chronic liver injury due to viral infections, alcohol intake or toxic agents can cause liver failure, cirrhosis and/or eventually lead to hepatocellular carcinoma [28]. Liver in general has good inherent regenerative capacity and the presence of hepatic progenitor cells helps restoration of liver structure and function [29,30]. However, this self-regeneration is true only in the early stages of liver disease, whereas in advanced stages the liver structure and function are maximally compromised and self-regeneration is poor [31]. Under such circumstances, the use of autologus/allogeneic stem cells or their derivatives may help liver regeneration [2, 32]. However, it is imperative to understand whether transplanted/delivered stem cells effectively reach their intended targets, integrate with the host tissue and contribute to the restoration of function. Towards this end, much recent advancement in cell tracking has been achieved.
In the present study, we utilized MSCs to alleviate cirrhosis in a rat model and we have successfully tracked the homing of the iron oxide (SPIONs) labelled MSCs using MRI. We also observed that homed MSCs helped reduction of the induced cirrhotic lesions as well as contributed to liver regeneration.
In the present study, the iron oxide nano particles cultured with BM-MSCs were readily internalized. This was facilitated by the presence of poly-L-lysine, which is well known to stimulate the cellular endocytosis of iron-oxide nano particles [21]. Furthermore, the iron oxide nano particles were inert and the BM-MSCs exhibited normal cell viability and proliferation, as reported earlier [33].   Measuring the size of a representative focal lesion in the liver and the progressive reduction in its diameter associated with stem cell therapy in the present study, confirms and correlates with the amelioration of hepatic pathology [38]. In addition, the elevated liver enzymes (ALP, ALT, AST and GGT) associated with induction of cirrhosis were restored to normal levels following BM-MSCs therapy, indicating improvement in liver function. In general, MSCs have known to exert their benefits either by undergoing differentiation in the host tissue or by a paracrine mechanism by release of cytokines/chemokines that help reduce the inflammation, fibrosis and oxidative stress associated with the disease [28]. Furthermore, MSCs are hypoimmunogenic in nature and both autologous and allogeneic MSCs from various sources have been used in the past for the treatment of liver related diseases with variable success [28, 46, 47]. More ongoing clinical trials indicate that stem cells will find increasing applications in liver diseases and therefore use of imaging techniqueswill allow effective monitoring of the administered stem cells and progress in disease management.

Conclusion:
Cell based therapies for liver diseases are rapidly increasing and some studies have progressed to Phase I clinical trials. Our study highlights the use of BM-MSCs in the amelioration of liver cirrhosis and how the stem cells can be labelled with SPIONs and tracked using MRI. The T2 weighted MRI sequences are best suited to evaluate liver cirrhosis/liver tumours and SPION labelled MSCs can be tracked in real time to confirm homing of transplanted cells and follow up with disease prognosis.