Magnetic Resonance Imaging of Ferumoxytol-Labeled Human Mesenchymal Stem Cells in the Mouse Brain

The success of stem cell therapy is highly dependent on accurate delivery of stem cells to the target site of interest. Possible ways to track the distribution of MSCs in vivo include the use of reporter genes or nanoparticles. The U.S. Food and Drug Administration (FDA) has approved ferumoxytol (Feraheme® [USA], Rienso® [UK]) as a treatment for iron deficiency anemia. Ferumoxytol is an ultrasmall superparamagnetic iron oxide nanoparticle (USPIO) that has recently been used to track the fate of transplanted cells using magnetic resonance imaging (MRI). The major objectives of this study were to demonstrate the feasibility of labeling hUCB-MSCs with ferumoxytol and to observe, through MRI, the engraftment of ferumoxytol-labeled human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) delivered via stereotactic injection into the hippocampi of a transgenic mouse model of familial Alzheimer’s disease (5XFAD). Ferumoxytol had no toxic effects on the viability or stemness of hUCB-MSCs when assessed in vitro. Through MRI, hypointense signals were discernible at the site where ferumoxytol-labeled human MSCs were injected. Iron-positive areas were also observed in the engrafted hippocampi. The results from this study support the use of nanoparticle labeling to monitor transplanted MSCs in real time as a follow-up for AD stem cell therapy in the clinical field.


Introduction
The curative and regenerative properties of stem cells have marked them as promising candidates as novel therapeutic agents for various neurodegenerative diseases [1][2][3]. Out of the wide range of neurodegenerative diseases, stem cell application seems to be particularly appealing for Alzheimer's disease (AD) [4][5][6]. Considered the most widely known and common type of dementia, the pathology of AD is complex and the causes are multifactorial [7,8]. With such complications present, mesenchymal stem cells (MSCs) have become a promising therapeutic agent for AD stem cell therapy because of the multiple functional roles that they can mediate through their paracrine activities [9][10][11]. The multiple roles of MSCs, ranging from anti-apoptosis to anti-inflammation, make them ideal for the treatment of heterogeneous diseases such as AD where they can target more than one mechanism [12][13][14][15]. Using a transgenic AD mouse model, our group has observed Electronic supplementary material The online version of this article (doi:10.1007/s12015-016-9694-0) contains supplementary material, which is available to authorized users. the removal of amyloid plaques and acceleration of endogenous neurogenesis through treatment with MSCs [16][17][18].
In order for these MSCs to exert their therapeutic benefits at the target site, the delivery route is of key concern [19]. Previously, our group has reported that MSC engraftment in the brains of transgenic AD mice is very low when cells are delivered through the intra-venous [20] and intra-arterial routes [21]. In contrast, intra-parenchymal administration is invasive, although maximal engraftment of MSCs can be achieved. In parallel to our pre-clinical studies [16,17,20], our group has also conducted a phase I clinical trial involving the intra-parenchymal administration of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) into the hippocampus and precuneus of AD patients [22]. Through this study, we were able to confirm the safety and feasibility of implanting cells directly into the brain parenchyma. However, one limitation was that the distribution of the transplanted cells could not be assessed through non-invasive means.
Cells can be observed non-invasively using two methods: a direct method that involves labeling stem cells with nanoparticles and an indirect imaging method that utilizes reporter genes [23,24]. Out of the various imaging modalities, magnetic resonance imaging (MRI) possesses several advantages that make it suitable for cellular imaging, such as superior soft tissue contrast and high resolution [25]. Cells can be labeled with iron oxide nanoparticles to perform MRI [26,27]. In 2009, the U.S. Food and Drug Administration (FDA) approved the use of ferumoxytol (Feraheme® [AMAG Pharmaceuticals, USA], Rienso® [Takeda Inc., UK]), an ultrasmall superparamagnetic iron oxide nanoparticle (USPIO), as a treatment for iron deficiency anemia in chronic kidney disease [28,29]. Ferumoxytol (off-label use) has been used widely in pre-clinical studies for cellular imaging purposes [29][30][31][32].
Utilizing these characteristics of ferumoxytol, the major objectives of this current study were first to investigate the feasibility of labeling hUCB-MSCs with ferumoxytol in vitro, and then to monitor in vivo engraftment of MSCs through MRI. To reduce the risk of engraftment failure, intra-parenchymal administration was chosen to noninvasively image MSCs that had been directly delivered into the hippocampus of the 5X familial Alzheimer's disease (FAD) mouse model.

Ethical Statement
This study was reviewed and approved by the Institutional Review Board (IRB) of Samsung Medical Center (IRB no. 2015-01-028). This research was also approved by the Institutional Animal Care and Use Committee (IACUC) of the Samsung Biomedical Research Institute (SBRI) at Samsung Medical Center (SMC). The SBRI is an accredited facility of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International) and abides by the Institute of Laboratory Animal Resources (ILAR) guide.
Cell Viability Assay hUCB-MSCs were initially seeded in six replicates of 96-well plates at a density of 9.6 × 10 3 per well for 24 h. MSCs were treated with 2 U/mL heparin and various doses of protamine sulfate and ferumoxytol for an additional 24 h. After the incubation period, cells were assayed for viability using the Alamar blue assay (Sigma-Aldrich, St. Louis, MO, USA). Cells were treated with the Alamar blue reagent for 3 h at 37°C and 5 % CO 2 , and fluorescence was read by a multiplate reader (GloMax®-Multi Detection System; Promega, Madison, WI, USA).

Prussian Blue Staining
Unlabeled and ferumoxytol-labeled hUCB-MSCs were washed with DPBS (Biowest) and then fixed with 4 % paraformaldehyde (Biosesang, Gyeonggi-do, Republic of Korea) for 15 min at room temperature (RT). Cells were washed with DPBS before staining. Paraffin blocks of the ferumoxytol-labeled hUCB-MSCs were prepared as described previously [21]. Staining was performed as instructed by the manufacturer (NovaUltra Prussian Blue Stain Kit; IHC WORLD, Woodstock, MD, USA). Stained slides were scanned using Aperio Scan Scope AT and visualized through the Aperio Image Scope program (Leica Biosystems, Buffalo Grove, IL, USA).

Trilineage Differentiation and Evaluation
Adipogenic differentiation was induced using the StemPro Adipogenesis Differentiation Kit (Thermo Fisher Scientific). hUCB-MSCs were labeled with ferumoxytol for 24 h in a 6well plate, washed three times with DPBS, and the media was replaced with the adipogenic base medium. The medium was changed twice a week for a total of 2 weeks. Cells were fixed with 4 % PFA and stained with Oil Red O (Sigma-Aldrich). To induce osteogenic differentiation, cells were first labeled with ferumoxytol as described above and then cultured in osteogenic base medium using the StemPro Osteogenesis Differentiation Kit (Thermo Fisher Scientific). The medium was changed twice a week for one week. After fixation using a solution containing citrate and acetone, mineralized matrix was assessed by alkaline phosphatase staining (Sigma-Aldrich).
Sections of the pellets were prepared at 5-μm thickness using a cryotome (Thermo Fisher Scientific) and stained with Safranin-O (Biosesang). Stained slides were observed using an inverted light microscope (U-HGLGPS, Olympus, Japan).

Experimental Animals
5XFAD transgenic AD mice that overexpress mutant human APP(695) with the Swedish (K670 N, M671 L), Florida (I716V), and London (V717I) familial Alzheimer's disease (FAD) mutations and human PS1 harboring two FAD mutations, M146 L and L286 V, were purchased from Jackson Laboratories (Bar Harbor, ME, USA). These transgenic mice were crossed, and the offspring were genotyped by DNA extraction from tail samples. These mice were fed ad libitum and were also maintained in a 12 h light/12 h dark cycle. A total of fifteen 7-8 month old mice were used for this study: sham (n = 3), MR imaging right after injection (n = 3), post 1 day (n = 3), 7 days (n = 3), and 14 days (n = 3).

Hippocampal Injections of Unlabeled and Ferumoxytol-Labeled hUCB-MSCs
After labeling with ferumoxytol for 24 h, hUCB-MSCs were washed three times: first with DPBS, second with DPBS containing heparin (10 U/mL), and finally with DPBS again. Cells were detached using 0.25 % Trypsin-EDTA (Gibco-Invitrogen). Ferumoxytol-labeled hUCB-MSCs were resuspended in phenol red-free MEMα1× media (Gibco-Invitrogen) prior to injection. Animals were stably positioned on a stereotactic device (Harvard Apparatus, Holliston, MA, USA) using ear bars. Coordinates of A/P − 1.94 mm, M/L ± 1.00 mm, and D/V − 2.6 mm were used to perform the sham operation, which only involved insertion and removal of a Hamilton syringe needle (Hamilton Company, Reno, NV) or injection of unlabeled (2 × 10 5 /3 μL), and/or ferumoxytol-labeled hUCB-MSCs (2 × 10 5 /3 μL) into the left and right hippocampus of 5XFAD mice, respectively, at a rate of 0.5 μl/min. Following the infusion, a 5 min delay time was provided before retracting the syringe needle. Mice were initially anesthetized with 5 % isoflurane (Hana Pharmaceutical Co., Ltd., Seoul, Republic of Korea) and subsequently maintained under 2 % isoflurane during the whole procedure.

Magnetic Resonance Imaging
A 7 T/20 MRI System (Bruker-Biospin, Fällanden, Switzerland) was used to acquire MR images of the phantom cell samples and the mouse brain. Unlabeled and ferumoxytol-labeled cells were mixed with 0.8 % agarose (Bioplus, Gyeonggi-do, Republic of Korea) to prepare the phantom samples. The tubes were filled with distilled water to reduce background artifacts. MR images were obtained using a multislice multiecho (MSME) spin echo sequence with the following parameters: repetition time (TR)/echo time (TE) = 2500/14-230 msec, slice thickness = 0.7 mm, number of averages = 4. MR images of the mouse brain were acquired as described previously [ 21]. T2-weighted images were obtained using the following parameters: TR/TE = 2500/20 msec, number of averages = 8, and slice thickness = 0.7 mm. A T2* gradient echo (GRE) sequence was used to acquire MR images using the following parameters: TR/TE = 303.805/18 msec, number of averages =8, slice thickness = 0.7 mm, and flip angle (FA) = 45°. Mice were anesthetized under 2 % isoflurane while the MR images were obtained.

Tissue Fixation and Immunohistochemistry
At the respective time points, mice underwent MR imaging and were sacrificed via cardiac perfusion. After fixation in 4 % PFA for 24 h, paraffin blocks were prepared, and 4-μm sections were cut using a microtome (Thermo Fisher Scientific). Sections were deparaffinized, and Prussian blue staining was conducted using the protocol described above. For immunohistochemical (IHC) staining, 1× citrate buffer, pH 6 (Dako, Carpinteria, CA, USA) was first used to perform heat-induced antigen retrieval. Immunofluorescence staining was performed according to our previous report [21]

Statistical Analysis
All data presented in the paper are expressed as mean ± standard error of mean (S.E.M.). A P value ≤0.05 was considered statistically significant. Student's t-test was used to investigate the differences between groups.

Characterization of Ferumoxytol-Labeled hUCB-MSCs in vitro
Using the heparin-protamine sulfate-ferumoxytol nanocomplex technique, blue staining was seen in the cytoplasm of hUCB-MSCs labeled with ferumoxytol compared with the unlabeled hUCB-MSCs (Fig. 1a). Differences in morphology of hUCB-MSCs such as adherent, bipolar, and fibroblast-like characteristics [35] were not noted following labeling (Fig. 1a). The efficiency of ferumoxytol labeling of hUCB-MSCs was further observed in the detached re-suspended state by creating cell paraffin blocks (Fig. 1b). Unlike the unlabeled hUCB-MSCs, brown staining was grossly observed from the pellet of ferumoxytollabeled hUCB-MSCs (Fig. 1c), a common observation for cells labeled with iron oxide nanoparticles [36,37]. The overall labeling efficiency was 85.84 ± 2.02 % (mean ± S.E.M.). To assess alterations in cell viability, hUCB-MSCs were treated with a constant dose of heparin and varying doses of protamine sulfate and ferumoxytol. The cell viability percentages after treatment with 10/100, 40/100, and 40/400 μg/mL of protamine sulfate/ ferumoxytol were 91.64 ± 3.66, 83.86 ± 4.58, and 84.19 ± 5.76 %, respectively (Fig. 1d). Labeling the cells did not affect the overall viability of the cells; however, increasing the concentration of protamine sulfate decreased the viability by approximately 20 %. A possible explanation is the toxic effects exerted by the positive charges of protamine sulfate [38]. However, the overall viability was still high probably due to the counteractive roles of heparin on protamine sulfate [38].

Differentiation Potential and Stemness of Ferumoxytol-Labeled hUCB-MSCs
Trilineage differentiation was not affected by labeling hUCB-MSCs with ferumoxytol. For both unlabeled (Fig. 2a) and ferumoxytol-labeled hUCB-MSCs (Fig. 2b), induction of adipogenesis was demonstrated by the accumulation of lipidrich vacuoles inside the cells that were stained with Oil red O (Fig. 2a, b). Osteogenic differentiation was observed in both unlabeled and ferumoxytol-labeled cells that expressed alkaline phosphatase (Fig. 2a, b). The expression of sulfated proteoglycans was detected by Safranin O staining (Fig. 2 a, b).

Detection of Hypointense Signals from Transplantation of Ferumoxytol-Labeled hUCB-MSCs in the Mouse Hippocampus
The in vivo MR characteristics of ferumoxytol-labeled hUCB-MSCs were preliminarily assessed by performing a sham surgery and intra-parenchymal administration of ferumoxytollabeled MSCs into the right hippocampus. MR images were acquired immediately after surgery. In the T2-weighted and T2* GRE MR images, the sham group displayed a hypointense (dark) vertical line extending downward from the cortex, representing the location of the needle track (Fig. 3a). However, the manifestation of the hypointense signal was clearly distinguishable from the group that received ferumoxytol-labeled hUCB-MSC injections, which showed hypointense signals that permeated or spread at the end of the needle track and created a diagonal linear (T2-weighted) or ovular (T2* GRE) appearance (Fig. 3b). The presence of ferumoxytol-labeled hUCB-MSCs was confirmed through Prussian blue staining (Fig. 3b).
Subsequently, engraftment of ferumoxytol-labeled hUCB-MSCs was observed at post 1 day. MR images acquired at 1 day post injection displayed dark, hypointense signals in the right hippocampus of 5XFAD mice where the ferumoxytol-labeled hUCB-MSCs were injected (Fig. 4a). Although a strong blooming effect was evident, the appearance of the signal was similar to that from MR images acquired immediately after the transplantation. Other than slight hyperintense signals showing the site of needle puncture in the T2-weighted MR images, no hypointense signals were detected in the left hippocampus, where unlabeled hUCB-MSCs were administered (Fig.4a).
Although the percentage of mitochondria-positive cells among the total cells (Hoechst stained) was slightly higher in the right hemisphere, the differences between the two hippocampi were not statistically significant (Fig. 4c). In addition to corroborating human MSC engraftment, iron-positive areas were stained blue in the right hippocampus by Prussian blue staining, whereas none were discernible in the left hippocampus (Fig. 4d). The absence of human cells in the dorsal 3rd ventricle (Fig. 5a) and lateral ventricles of the left and right hemispheres (Fig. 5b, c) confirmed the accuracy of the engraftment and suggested that the cells did not migrate into ventricular regions by possible back flow that may arise along the needle track.

No Infiltration of Inflammatory Cells into the Site of MSC Engraftment
Additional IHC staining using anti-Iba-1 (microglia/macrophage) and anti-GFAP (astrocyte) antibodies was performed to evaluate inflammatory effects that may arise from injection of unlabeled/ferumoxytol-labeled hUCB-MSCs and to corroborate the source of the MR signal. Based on microscopic evaluations, a dramatic difference in Iba-1 and GFAP expression was not evident between the left and right hippocampus (Fig. 6a). Interestingly, very low or no expression of Iba-1 or GFAP was apparent at the site of hUCB-MSC engraftment (indicated with a white dotted line) in the right hippocampus, which was confirmed by staining an adjacent slide using the anti-mitochondria antibody (Fig. 6b). These observations were quantitatively assessed, and the percentages of Iba-1 and GFAP-positive cells over the total number of cells (Hoechst) were determined for both the left and right hippocampi (Fig. 6c). Compared to the left hemisphere, the percentages of Iba-1 and GFAP were lower or similar, respectively, in the right hippocampus. Although not investigated in detail, the slightly higher engraftment percentage of MSCs in the right hippocampus could have exerted more immunomodulatory effects to contribute to the lower Iba-1 percentage in comparison to the left hippocampus.

Persistence of Hypointense Signals at Prolonged Time Points
With such observations made at post 1 day, a question that arose was whether hypointense signals will be observed at the Fig. 3 Differences in Hypointensities Generated from Sham-Operated and Ferumoxytol-labeled hUCB-MSC Injected Mice. a A linear hypointense signal from T2-weighted and T2* GRE images revealed the puncture site and needle track. b In contrast, diffusion of hypointense signals was discernible in the right hippocampus of mice that received injections of ferumoxytol-labeled hUCB-MSCs. Prussian blue stains revealed iron positive cells in the right hippocampus. Scale bar: 80 μm engraftment site past 1 day. Therefore, additional experiments were conducted under the same experimental settings as the post 1 day group, and the mice were monitored at 2 time points: post 7 and 14 days. For both groups, at both post 7 days, the hypointense signal was consistently observed at the right hippocampus (Fig. 7a, b). Especially discernible from the T2* GRE images, the overall size of the signal was decreased, when compared to the image acquired at post 1 day   (Fig. 7a, b). The size and appearance of the signal detected at post 14 days resembled that observed at post 7 days (Fig. 7b).

Discussion
In this study, ferumoxytol-labeled hUCB-MSCs that were engrafted in the hippocampus of 5XFAD transgenic AD mice were observed using MRI with the hope of supporting the application of this technique in AD stem cell therapy. Although invasive brain penetration and confined cellular distribution are disadvantages of stereotactic delivery, the method is still a well-established technique that has been widely used in small animals and allows for localization of cells at the target site [40]. In our study, we targeted the hippocampi of 5XFAD transgenic mice that are known to develop high amyloid deposition at very early stages of disease progression [41]. Considering these pathological features and the presence of the blood brain barrier (BBB), intra-parenchymal administration is preferred in the clinical settings; our group has clinically confirmed the safety and feasibility of delivering stem cells by utilizing this route in AD patients [22].
The first goal of this study was to demonstrate the feasibility of labeling hUCB-MSCs with ferumoxytol. Our results were in agreement with past studies that supported the use of the heparin-protamine sulfate-ferumoxytol nanocomplex as an efficient labeling method [29,30,34]. We observed in vitro ferumoxytol labeling of hUCB-MSCs in both adhered and detached states. Iron-positive staining of detached ferumoxytol-labeled hUCB-MSCs confirmed the stability of ferumoxytol labeling even after multiple washings, centrifugation, and re-suspension. In addition to efficient labeling, ferumoxytol labeling did not negatively influence the expression of certain cell surface markers or the differentiation capabilities of hUCB-MSCs in vitro. The fibroblastic characteristics and overall proliferation of the labeled cells were not impaired [42,43]. There are evidences of MSCs differentiating into ectodermal lineages if pre-induced or introduced to developmental cues in vivo [44,45], but examining such potentials was not defined as the major objectives of this present study. The second goal of this study was to observe the engraftment of ferumoxytol-labeled hUCB-MSCs at the target site. As expected, iron-positive hUCB-MSCs were accurately delivered and engrafted into the mouse hippocampus. Growth of the transplanted cells was not examined in depth because MSCs have a low probability of proliferating in vivo, which is probably why they do not generate teratomas unlike embryonic and induced pluripotent stem cells [46,47]. Considering the aggregated state of the cells, limited distribution seem to have occurred for both unlabeled and ferumoxytol-labeled hUCB-MSCs. Compared to the left hippocampus (unlabeled hUCB-MSCs as a control), a prominent negative contrast was discernible from the right hippocampus where ferumoxytollabeled hUCB-MSCs were injected. Such hypointense signals were visualized at various time points from immediately to post 14 days following stereotactic delivery. In contrast to the T2-weighted images, the enlarged areas of dark signals in the T2* GRE images are representative of the blooming effect or susceptibility artifacts which decreased with time [48]. While T2 weighted spin echo lacks iron sensitivity, T2* is much more sensitive but does not provide a reliable representation of the engraftment size. Thus, the two sequences compensate for each other's weaknesses and were both used to noninvasively image the engraftment site.
Other than the blooming effect, alternative confounding factors that may restrict the interpretation of MR images include (but are not limited to) brain puncture, hemorrhage, and activation of macrophages. First, signs of hemorrhages were not recognized from MR images, which excluded bleeding as a source of the negative signal. Based on the images of the sham group, the hypointense signal was generated from penetration of the needle into the brain parenchyma and clearly differed from that created from the injection of ferumoxytollabeled hUCB-MSCs [49,50]. An interesting observation was that infiltration of inflammatory cells was not detected at the sites of engraftment for either unlabeled or ferumoxytollabeled hUCB-MSCs. This indicated that macrophage engulfment of iron and/or dead cells were not the origin of the hypointensity [51]. However, former groups have proposed that SPIO-labeled cells underwent apoptosis and phagocytosis by macrophages when observed as early as 1 to 2 weeks post transplantation [51][52][53][54]. Considering such reports, we cannot rule out the possibility that apoptosis and inflammatory cell infiltration might be apparent as was as reduced cell survival at the site of ferumoxytol-labeled hUCB-MSC engraftment if monitored at later time points past 1 day.
This study has several limitations that must be discussed. First, as stated above, cell fate was not evaluated at time points past one day. However the gradual decrease in hypointensity observed over time is suggested to be indicative of cell survival [26]. Thus, we expect the residual, viable cells from the initial transplantation to contribute to the generation of the hypointense signal. Second, the efficacy of the ferumoxytol-labeled hUCB-MSCs in the transgenic AD mouse model was not examined in depth. The major aims of our study were to assess the feasibility of labeling hUCB-MSCs with ferumoxytol and their detection using MRI. As a result, potential therapeutic benefits or biological roles of ferumoxytollabeled hUCB-MSCs in the host environment were not extensively investigated. Since the ferumoxytol-labeled hUCB-MSCs met the criteria proposed by the ISCT, we presume that the labeled cells would not fall short of the stemness, including immunophenotype and therapeutic potential, displayed by unlabeled, naïve MSCs. Further study is warranted to evaluate the efficacy of ferumoxytol-labeled hUCB-MSCs, their survival, and its correlation to the persistence of negative signals detected from MRI.
In summary, the results from the current study introduce use of the heparin-protamine sulfate-ferumoxytol nanocomplex to label hUCB-MSCs and show that labeled cells can be detected noninvasively using MRI following transplantation. We hope that this method will be applied as a follow-up for clinical cell transplantations not only in AD, but also in various other diseases.