Monitoring ferumoxide-labelled neural progenitor cells and lesion evolution by magnetic resonance imaging in a model of cell transplantation in cerebral ischaemia

Neural stem cell cultures

For initial cell labelling assays and quantification of iron, the ST14A rat neural progenitor cell (NPC) line was used, and cultured as previously described (generous gift from E. Cattaneo)²⁶. For subsequent labelling, viability, proliferation and differentiation assays, and for the in vivo study, primary embryonic forebrain-derived NPCs were used.

Forebrain tissue was dissected from E13.5 CD1 mouse embryos (Charles Rivers UK) and digested in Trypsin with DNase. Cells were washed in trypsin inhibitor and Earl’s Balanced Salt Solution (EBSS), and plated in 6 well culture dishes. Proliferation medium contained DMEM:F12 with glutamax (Invitrogen), N2 supplement (GIBCO), 10 ng/ml EGF (Peprotech), 20 ng/ml FGF-2 (Peprotech), 0.05% heparin and 1% Penicillin-Streptomycin. Cells were incubated at 37°C with 5% CO₂. Under these conditions neural stem/progenitor cultures (NPC) form aggregates called neurospheres that grow in suspension. Neurospheres were passaged every 7 days. The ST14A rat neural progenitor cell line was cultured as previously described²⁶. All reagents were from Sigma, unless otherwise stated.

Cell labelling with contrast agent

For neurosphere labelling, neurospheres were dissociated at 6 days after passage, by incubation in 1X trypsin with DNase for 7 minutes at 37°C. Cells were washed in trypsin inhibitor and EBSS, and then incubated with contrast agent (Endorem or the FePro complex) in proliferation medium for 24 hours to allow contrast agent internalisation. Cells were washed in EBSS, and dissociated for replating or use in intracerebral microinjections.

For adherent ST14A cell labelling, cells were incubated with contrast agent in culture medium for 24 hours to allow contrast agent internalisation, washed three times with EBSS, then passaged.

Endorem: Cells were incubated with 500 µg/ml Endorem (Guerbet Laboratories Ltd, UK).

FePro complex: 2 mg/ml protamine sulphate was incubated with Endorem in a ratio of 9:5 for 10 minutes at room temperature to form the complex (FePro), making a final concentration of 100 µg/ml iron and 10 µg/ml protamine sulphate in the culture medium.

Cellular viability assays

Cells were incubated with Endorem, FePro, or no contrast agent (control) for 24 hours. Cells were washed and replated into 96 well plates at a density of 10⁴ cells per well, 8 wells per treatment group, for cell metabolic activity assays as a measure of cell viability. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; Sigma) and Alamar Blue (Invitrogen) assays were performed as previously described^27,28. For the Trypan Blue assay, cells were resuspended 10:1 in 0.5% Trypan Blue (Sigma), and transferred to a haemocytometer for counting. Under a light microscope (Zeiss), non-viable blue-stained cells and total viable cells were counted. For each assay, two samples were counted for each treatment group, and the assay was repeated three times.

Cellular proliferation assays

The neurosphere-forming assays measured the number of neurospheres generated per 1000 cells and the average neurosphere diameter per sample, which are indicators of the number of stem and progenitor cells in the sample and their rate of proliferation, respectively. Cells were plated at a density of 10⁴ cells per ml in neurosphere proliferation medium into 96 well plates, where each well contained 100 µl of proliferation medium and 10³ cells. After 5 days in culture, the number of neurospheres generated per 10³ cells was counted, and neurosphere diameter was measured using the imaging analysis software, ImageJ developed by U. S. National Institutes of Health, Bethesda, Maryland, USA (http://imagej.nih.gov/ij/, 1997–2012). For each assay, a minimum of 8 wells per treatment group was counted, and each assay was repeated twice.

Prussian blue stain for iron in neurospheres

NPCs cultured at a density of 10⁴ cells per ml for 7 days were transferred to polyornithine-coated coverslips in 24 well plates for one hour to allow neurosphere adherence to coverslips. NPC were then fixed in 4% paraformaldehyde (PFA) in PBS for 10 minutes, incubated in 6% hydrochloric acid and 4% potassium ferrocyanide for 20 minutes, then washed in PBS and mounted.

Cellular differentiation assays

Neurospheres at 6 days after passage were differentiated as previously described²⁹. Differentiated neurospheres were fixed in 4% PFA at 4°C. For immunocytochemistry, coverslips were incubated in blocking solution (10% goat serum with 0.1% Triton-X) for 30 minutes, then incubated in primary antibody in blocking solution for one hour at 37°C: IgG₁ monoclonal mouse anti-β3-tubulin (1:1000, Promega G7121: G-purified monoclonal antibody from clone 5G8 raised in mice against a peptide (EAQGPK) corresponding to the C-terminus of β3-tubulin; the antibody has been tested to perform in frozen and paraffin-embedded sections of rat brain, cerebellum and spinal cord, human and rat fetal CNS progenitor cell cultures and adult human paraffin-embedded brain); polyclonal rabbit anti-GFAP (1:1000, Chemicon AB5804; raised in rabbit against purified bovine GFAP; routinely evaluated by immunohistochemistry on brain tissue, astrocytes, and neurons by Chemicon); monoclonal O4 IgM (1:2, generous gift Prof Rhona Mirsky)^30,31. Coverslips were washed three times in PBS, then incubated with secondary antibody for 30 minutes at 37°C. Secondary antibodies were: Cy3-conjugated goat anti-mouse (1:100, Invitrogen A10521); Alexa 488-conjugated goat anti-rabbit 1gG (1:1000, Invitrogen A11008); and Alexa 680-conjugated goat anti-mouse IgM (1:1000, Invitrogen A21048). Coverslips were washed in PBS and mounted onto slides for fluorescence imaging.

Measurement of iron using a superconducting quantum interference device (SQUID)

ST14A cells were labelled with Endorem or FePro using the labelling methods described above. Labelled cells were then washed and fixed in 4% PFA. Cells were transferred to gelatin capsules (size 0, Capsugel) containing cotton wool, at a density of 1.25 × 10⁶ per gel cap. Iron content of samples was measured using a superconducting quantum interference device (SQUID) and the amount of iron per cell was calculated. Sample magnetization was measured over a range of magnetic field strengths at 300K (Kelvin; room temperature) and 10K, and hysteresis loops were plotted. To calculate the amount of iron in each sample, remnant magnetization and saturation magnetization of the samples were compared to the magnetization of a sample with Endorem alone, which had a known iron content against which cell magnetisation was calibrated.

Middle cerebral artery (MCA) occlusion and intracerebral microinjections

All procedures were carried out under the Animals Scientific Procedures Act 1986 (project license: PPL 70/05617). Eleven wild type male Sprague-Dawley rats (250 g, 8 weeks old, Charles Rivers, UK; n=11) were anaesthetised with 2% isofluorane. Temperature was monitored with a rectal probe. The middle cerebral artery occlusion was performed as previously described³², to occlude the MCA for 30 minutes. Briefly, the right common carotid artery was exposed. A sterile 4-0 suture with an epoxy resin tip (Araldite^®, Huntsman Advanced Materials) was inserted into the carotid artery and advanced 17 mm into the brain to occlude the MCA for 30 minutes. Following occlusion, the MCA was reperfused by slowly removing the suture, and animals were then recovered on a heated mat. Animal weight was monitored. Following MCAO surgery, animals were assigned a number and alternately allocated to control or treatment group. After surgery, animals were housed individually, provided with soft tissue bedding and had unrestricted access to water and softened food pellets.

In six animals, FePro-labelled NPCs (FePro-NPCs) were injected into the ipsilateral corpus callosum 48 hours following cerebral ischaemia. Animals were anaesthetised with 2% isofluorane, and secured in a stereotactic frame (Kopf, Germany). Animals were injected with 1.25 × 10⁵ FePro-labelled cells in a maximum of 3 µl, as described above, at 0.2 l/min into the ipsilateral corpus callosum. The coordinates used were AP +1.0, ML -0.3; DV -0.2 to Bregma. Five animals received no intracerebral injection (control uninjected group).

Animals were imaged to determine whether injected cells could be identified and monitored over time with MRI, relative to the control uninjected group.

MRI

For in vitro MRI, contrast agent-labelled cells were washed and fixed in 4% PFA. Cells were transferred to 250 µl Eppendorf tubes and centrifuged to produce a cell pellet. Eppendorfs were placed in a custom-made probe and imaged using a 2.35T horizontal bore magnet (Oxford Instruments, UK) interfaced to an SMIS console (Guildford, UK). A 2D spin echo sequence was used with the following parameters: TR=1500 ms; TE=80 ms; FOV 25 mm; slice thickness 1 mm.

For in vivo MRI, animals were anaesthetised with 2% isofluorane, and secured on a stereotactic probe. Animals were imaged using the above 2.35T system. A 2D gradient echo (T₂*-weighted) sequence was used with the following parameters: TR=500 ms; TE=30 ms; FOV 30 mm; 128 × 128 voxels; 11 slices; 1 mm slice thickness; and 16 averages. A 2D spin echo (T₂-weighted) sequence was also used: TR=1500 ms; TE=120 ms; FOV 30 mm; 128 × 128 voxels; 7 slices; slice thickness 1 mm; 16 averages. Animals were imaged at 3, 7, 14, 21, and 28 days post MCA occlusion surgery. For lesion volume measurements, hyperintense lesion areas from T₂-weighted images were segmented manually using ImageJ software available at http://rsb.info.nih.gov/ij (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD). Lesion areas per slice were used to determine the total brain lesion volume, taking into account the thickness of the 2D slices (total brain lesions covered a mean of 7.33 (1.44 SD) slices in FePro-NPC animals and 7.50 (1.27 SD) slices in control animals). The lesion volume of the central slice covering the central MCA territory was measured and compared between groups. Homologous regions of interest were defined in the contralateral hemisphere.

Histology

After the final MRI time point, animals were euthanized by transcardial perfusion with 0.9% saline then perfusion fixed with 4% PFA. Brains were removed from the skull and transferred to 30% sucrose in PBS for 48 hours, then frozen and stored at -80°C for tissue processing.

Coronal brain sections of 30 μm thickness were taken on a cryostat (Bright Instruments, UK). For Prussian blue staining, sections were incubated in 6% hydrochloric acid and 4% potassium ferrocyanide for 20 minutes, then washed in PBS. Brain sections were counterstained with nuclear fast red or hematoxylin-eosin, and mounted with DPX (distyrene, plasticizer, xylene) mountant.

Neurosphere migration assay

The MCA occlusion surgery was performed and animals were imaged at 24 hours post-ischaemia to visualize the infarcted area. Four animals underwent MCA occlusion surgery and were euthanised using Schedule 1 method at 24 hours (n=2) or 48 hours (n=2) post-ischaemia. The area of infarct, as determined by MRI at 24 hours post-ischaemia, was isolated. The contralateral hemisphere was used as non-ischaemic control tissue. Tissue was homogenized and filtered through a 0.4 μm sterile filter. Protein content of the homogenate was calculated against an albumin standard using the Pierce BCA Protein Assay Kit.

Single neurospheres were transferred into polyornithine-coated wells of 96 well plates, in 100 μl of culture medium per well. Individual neurospheres were photographed on a Zeiss AxioVert inverted microscope with a Hamamatsu digital camera and using OpenLab imaging software, then tissue homogenate or growth factors were added to the control medium. Treatment groups were as follows: 1400, 700, 300 μg/ml MCAO homogenate from 24 hours time point; 1400, 700, 300 μg/ml MCAO homogenate from 48 hours time point. Control groups were as follows: DMEM:F12 with N2 medium; DMEM:F12 with control tissue homogenate. Neurospheres were photographed 72 hours following treatment and the area covered by the cells measured to quantify the extent of migration. Assays were repeated three times, with 8 wells per treatment group.

Data analysis

Data are expressed as mean +/- standard error. Data analysis was performed using ImageJ for MRI image analysis and neurosphere diameter measurements; Adobe Photoshop for microscopy image analysis, and SPSS software for statistical analysis. A one-way ANOVA was used for statistical comparisons, with P<0.05 defined as significant. A two-way mixed ANOVA was used for statistical analysis of the change in lesion volume over time in control and FePro-NPC-treated animals.

Neurosphere response to cerebral ischaemia

We assessed whether the NPCs expanded as neurospheres from embryonic mouse forebrain were able to respond to cues from cerebral ischaemia. We investigated their migratory response to protein extracted from cerebral ischaemia infarct regions at two different timepoints – 24 and 48 hours post-ischaemia and from the corresponding contralateral control (Figure 1). The protein extracts were added directly to the wells containing the individual neurospheres; hence migratory cues were evenly distributed.

Figure 1. NPC neurosphere response to cerebral ischaemia.

A,B, Migration of individual neurosphere in response to 1400 μg/ml protein from 24 hour MCAO homogenate at t=0 hr (A) and t=72 hr (B). C,D, Migration of individual neurosphere in response to contralateral hemisphere MCAO proteins at t=0 hr (C) and t=72 hr (D). E, Cell migration from E14 neurospheres at 72 hours with no proteins or treatment with proteins from either normal brain homogenates or from 24 (light blue bars) and 48 (dark blue bars) hours MCAO homogenates at different concentrations; *: p<0.001. Scale bar, 400 µm.

As shown in Figure 1A–E, extensive migration in all directions had occurred by 72 hours from neurospheres exposed to ischaemia protein extracts (Figure 1A,B) but not in the presence of control proteins (Figure 1C,D). Figure 1E demonstrates the migratory response of NPC to 24 hr and 48 hours ischaemic tissue protein extracts at different protein concentrations. Migration in both 24 hours and 48 hours post-ischaemia groups was significantly different from control migration (F=6.680; p<0.001). Post hoc analysis showed that at 24 hr, the 1400 µg/ml concentration, and at 48 hours the 300 and 700 μg/ml MCAO concentrations, were significantly different from control non-ischaemic tissue protein extract. Hence NPCs are able to respond to cerebral ischaemia cues in vitro.

FePro cell labelling and viability

The effects of iron oxide-based MRI contrast agents on cell viability were first assessed in a fetal neural stem cell line and on primary NPCs. Incubation of ST14A cells with Endorem or FePro for 24 hours did not affect cell viability as assessed using metabolic activity assays (Supplementary Figure 1). Iron content was measured using a SQUID. Mean iron uptake was 3.93 pg Fe/cell for Endorem-labelled cells, and 14.5 pg Fe/cell for FePro-labelled cells. Therefore the iron content was 3.7-fold greater in FePro-labelled cells than in Endorem-labelled cells. The efficiency of uptake of the FePro label into cells was not precisely quantified using Prussian Blue staining method, however high FePro accumulation was easily observed in at least 50% of the cells incubated with the contrast agent.

We then investigated the effect of cell labelling with FePro and Endorem on NPC viability and proliferative capacity (Figure 2A–D). Neither contrast agent affected NPC viability as shown by the Trypan Blue exclusion assay and Alamar blue assay (Figure 2A,B). Furthermore, neither neurosphere-forming ability nor neurosphere growth were negatively affected by FePro or Endorem (Figure 2C,D).

Figure 2. Effect of FePro- and Endorem-labelling on NPC viability and growth.

A, Alamar Blue assay. No significant difference in NPC metabolism (F=0.736, p=0.49) is observed. B, Trypan Blue assay. No significant difference in cell survival (F=0.106, p=0.901) is observed. C, Neurosphere diameter. Diameter (µm) of Endorem-labelled neurospheres is significantly different from control unlabelled neurospheres (F=7.497; p<0.05). D, Neurosphere forming ability. No significant difference in neurosphere formation per 1000 cells (F=0.184; p=0.833) is observed. E, F. Prussian Blue stain for iron in Endorem (G) and FePro (H) labelled neurospheres after 7 days in culture.

The iron within NPC neurospheres was visualized after 7 days in culture by Prussian blue. As shown in Figure 2E,F, the contrast agent is retained within cells across cell divisions.

To further characterise the effects of Endorem and FePro, the differentiation capacity of contrast agent-labelled NPCs was assessed. Figure 3 shows that FePro and Endorem-labelled NPCs can differentiate into neurons, oligodendrocytes, and astrocytes (Figure 3A–F). The cells retain the contrast agent label after differentiation (Figure 3G,H) and FePro-labelled cells generate negative contrast on MRI (Figure 3I,J).

Figure 3. Differentiation capacity of FePro-labelled NPC.

A–F. Immunocytochemistry of FePro (A–C) and Endorem (D–F) labelled neurospheres. A,D, anti-β3-tubulin (neuron, red). B,E, anti-GFAP (astrocyte, green). C,F, anti-O4 (oligodendrocyte, red). Blue, Hoescht nuclear stain. FePro-labelled neurospheres can differentiate into neurons (A,D), astrocytes (B,E), and oligodendrocytes (C,F). G, immunocytochemistry of FePro-labelled astrocyte (GFAP, green). H, corresponding light micrograph showing Prussian blue stain. I,J MRI T₂-weighted image of FePro-labelled neurosphere pellet (I) and control unlabelled cell pellet (J). FePro-labelled cell pellets produce hypointensity on MRI images. Arrows denote a GFAP-labelled cell co-labelled with Prussian blue, indicating that the FePro-labelled cell was capable of astrocyte differentiation. Scale bars, 20 µm.

Serial MRI of cerebral ischaemia following FePro-NPC injection

In vivo MRI of injected FePro-labelled NPC was carried out 3, 7, 14, 21 and 28 days after cerebral ischaemia, to determine whether cells could be identified and monitored over time, relative to a control MCAO group without NPCs (Figure 4). The T₂-weighted images revealed extensive heterogeneous ischaemic lesions in both groups, with hypointense regions developing in the striatum and middle cortical layers, from day 7 and persisting until day 28 (Figure 4A,B). This lesion heterogeneity was present in all control animals as well as in the FePro-NPC group. The FePro-labelled cells at the injection site were clearly detected in T₂*-weighted images (Figure 4C,D).

Figure 4. In vivo MRI.

A,B, Serial T₂-weighted imaging up to 28 days post-ischaemia in FePro-NPC and control animals. Regions of T₂ hypointensity develop in the lesion at later timepoints (white arrows). C,D, Serial T₂*-weighted imaging up to 28 days post-ischaemia in FePro-NPC and control animals. The injection site of FePro-labelled cells can be identified in the FePro-NPC group at all timepoints (black arrows).

We compared T₂- and T₂*-weighted images at 28 days in the FePro-NPC and control group. Figure 5A–D shows the profile of a FePro-NPC-treated animal and control animal at the 28 day time period. Lesion heterogeneity was observed in all animals in both T₂ and T₂*-weighted images. Signal intensity in hypointense ischaemic striatum regions was significantly different from the intensity in corresponding contralateral regions (Figure 5E; F=3.154, p=0.05).

Figure 5. Hypointensity at 28 days post-ischaemia.

T₂- (B,D) and T₂*-weighted (A,C) images are shown. A,B, MCA region in one control. C,D. MCA region in one FePro-NPC animal at 28 days post-ischaemia. Hypointense regions within the ischaemic lesion (arrows) were observed in both T₂- and T₂*-weighted images. E, Signal intensity in regions of T₂-weighted hypointense ipsilateral striatum at 28 days post-ischaemia, and in corresponding contralateral regions in control (green) and FePro-NPC (blue) groups. Signal intensity in the ischaemic striatum was lower than in the contralateral striatum. Signal intensity was significantly different between contralateral and ipsilateral hemispheres (p<0.001).

We investigated the origin of the hypointense signal on T₂-weighted MRI by examining histological sections. Iron can be a source of T₂-weighted hypointensity, and we assessed its distribution in both groups. In both groups, iron was detected in the ipsilateral (ischaemic) striatum at the ischaemic border, and in the ischaemic cortex in some animals (2 of 5 control, and 3 of 6 FePro-NPC animals)(Figure 6C–F). Iron accumulation was detected in the mid-striatum at the lesion border, which approximates the area of hypointensity on MRI. Additionally in the FePro-NSC group, iron was detected at the injection site (Figure 6G). No iron was detected in the contralateral cortex or striatum in the FePro-NSC group (Figure 6H).

Figure 6. Histological analysis at 28 days at the ipsilateral striatum.

A,B, Prussian blue stain for iron and hematoxylin-eosin staining at the ipsilateral striatum. Iron detected in the ischaemic lesion. C,D, Prussian blue stain with nuclear fast red counterstain in the ipsilateral striatum. E,F, OX-42 immunohistochemistry stain for macrophage/microglia, and Prussian blue stain for iron. Prussian blue-positive and – negative macrophage/microglia, detected by OX-42, are present at the ischaemic lesion. G, Prussian blue stain at the injection site in FePro-NPC ipsilateral hemisphere. H, OX-42 immunohistochemistry for macrophage/microglia and Prussian blue stain in the contralateral hemisphere. No macrophage/microglia or iron were observed in the striatum or cortex of the contralateral hemisphere. Scale bars, 30 µm.

We investigated whether the MRI signal hypointensity was associated with the distribution of macrophage/microglia in the brain. We observed accumulated macrophage/microglia in the infarcted cortex and striatum in both FePro-NSC and control groups, and some of them co-labelled with iron (Figure 6E,F). Iron-positive cells were a mixture of macrophage and non-macrophage cell types. No macrophage/microglia were observed in the contralateral hemispheres (Figure 6G,H).

Finally to investigate the effect of cell injections on the outcome of cerebral ischaemia, MRI analysis was carried out. Changes in volume of the lesion at the level of the central MCA region and the volume of the whole lesion over time were compared between control ischaemic and FePro-NPC groups. No statistically significant difference in lesion volume over time was observed either for the whole lesion or at the central MCA territory (Figure 7A,B; p=0.275 and p=0.244, respectively). The change in hemisphere volume at the MCA territory was also assessed. We observed that the ipsilateral hemisphere volume was decreased by 28 days post-ischaemia relative to the contralateral hemisphere in both groups (F=17.18; p<0.005; Figure 7C), but we did not observe a difference between FePro-NPC and control groups. This reduction in ipsilateral hemisphere volume over time may represent secondary, delayed neuronal death in the ischaemic hemisphere, lesion compaction, brain reorganisation or a combination of these factors.

Figure 7. Analysis of lesion volume change 7 days and 28 days after MCAO in FePro-NPC grafted and controls.

A, Lesion volume (cm³/mL) of the central MCA region slice. There is no significant difference between controls (solid line, blue) and FePro-NPC (dashed line, green) lesion volumes at the MCA region (p=0.577). B, Total lesion volume. There is no significant difference between control and FePro-NPC lesion volumes (p=0.921). C, Hemisphere volume change. Change in ipsilateral:contralateral hemisphere volume ratio between 7 and 28 days (*:ANOVA, p<0.0001), but there is no significant difference between control and FePro-NPC in hemisphere volume.

FePro displays high labelling efficiency and does not affect NPC behaviour

We observed that NPC can be stably labelled with FePro with no significant effect on cell viability, metabolic activity or proliferation relative to control, unlabelled cells, and that labelled NPC can differentiate into neurons, oligodendrocytes and astrocytes. The FePro label is retained throughout multiple cell divisions during the formation of new neurospheres and their differentiation. This is consistent with our previous in vivo labelling and tracking of endogenous stem cells that showed the presence of doublecortin-FePro-labelled cells 28 days after FePro injection into the lateral ventricle²⁴. In addition, data from the ST14A cell line (Supplementary Figure 1) also confirm that FePro does not affect cell viability, and has greater labelling efficiency than Endorem. Imaging NPC in vitro demonstrated that FePro uptake under the described labelling conditions was sufficient to generate MRI contrast. SQUID measurement of iron oxide uptake also confirmed and quantified cellular uptake of FePro and suggest that FePro may be a superior MRI contrast agent for NPC labelling compared with Endorem²⁴. Our results indicate that there are no cytotoxic effects associated with FePro labelling, which is consistent with other studies that have similarly shown limited cytotoxicity of FePro in other cell types, and that FePro uptake is sufficient to generate contrast on T₂*-weighted images^20,21,33,34. Taken together with our previous work demonstrating that endogenous neural stem cells could be labelled with FePro and migrate from the subventricular zone (SVZ) to the olfactory bulb²², NPCs are ideally suited for labelling with FePro for in vivo detection of cell migration.

Evolution of MRI T₂ contrast and heterogeneity interferes with NPC detection in ischaemic brain

This study demonstrated that cellular imaging was in part complicated by the evolution of T₂ and T₂* heterogeneity in the ischaemic lesion in all animals, obscuring cell detection on MRI at later timepoints. The evolution of T₂-weighted hypointensity could be related to the accumulation of macrophages and granulocytes to the lesion over time, leading to localized hypercellularity. Accumulation of MHC Class II-expressing cells (such as macrophages, dendritic and B cells) was shown to be spatially coincident with regions of T₂-weighted hypointensity and reduced ADC (apparent diffusion coefficient) in a model of multiple sclerosis³⁵. Hypercellularity was shown to increase from one week, peak at 3 weeks and persist in later timepoints, and regions of T₂-weighted hypointensity develop by 3 weeks, which is consistent with our results³⁵. In another study, T₂ hypointensity was observed from 4 days post-ischaemia at the lesion border, which interfered with detection of SPIO-labelled injected cells, and accumulation of ferric iron was detected histologically from 6 days post-ischaemia around the lesion³⁶. Although we did not observe haemorrhage in any of the animals, the T₂ heterogeneity may also arise as a consequence of the accumulation of iron in the lesion, through the development of microhaemorrhages and deposition of haemosiderin³⁷, the degradation of blood products, or neuronal degeneration.

In the FePro-NSC group, an additional factor is clearly the presence of iron oxide particles in the FePro contrast agent. Palweczyk et al. have shown that iron exchange can occur between labelled cells and macrophages in vitro, although the transfer of iron to macrophages is low³⁸. Weber et al. observed the evolution of hypointensity in ischaemic striatum on T₂*-weighted MRI at 2 and 10 weeks post-ischaemia in a MCA occlusion model, and found that T₂* hypointense regions colocalised with iron-containing macrophages³⁹. The authors attributed the iron in macrophages to the phagocytosis of red blood cells from damaged blood vessels. Danielisova et al. have observed iron deposition in the striatum and pyramidal cells of cortical layers III and V following 20 minute MCA occlusion⁴⁰. Justicia et al. have reported iron accumulation, T₂-weighted hypointensity and delayed neuronal death in the thalamus between 3 and 7 weeks following cerebral ischaemia⁴¹ and suggested that iron accumulation was mediated by heme oxygenase-1-positive (HO-1) microglial activity. These studies suggest that iron accumulation at lesion sites may be linked to microglial activity and neurodegeneration.

Whereas our previous study has shown in normal brains a good correlation between MRI and FePro-labelled endogenous neural progenitor cells²⁴, tracking FePro-labelled cells in the ischaemic brain is clearly more challenging. There are a few studies that have transplanted iron-oxide or gadolinium labeled ES or NSC cells into the contralateral, non-ischaemic hemisphere (rather than in the ipsilateral stroked hemisphere), and observed migration of cells towards the ischaemic lesion^19,42–44. In these studies, transplanted cells were capable of migration via the corpus callosum into the ishaemic hemisphere, and could be visualised using MRI. However, it should be noted that T2 and T2* heterogeneity in the ischaemic lesion evolves over time, and it clearly becomes an issue particularly at later time points. This study highlights the need to characterise the long-term profile of cerebral ischaemia, and points to confounding image contrast at later timepoints that must be overcome in order for iron oxide-based cellular imaging to be a viable method of cell tracking.

Soluble cues from injured tissue promote NPC migration in vitro

The NPC demonstrated a migratory response to cerebral ischaemia cues in vitro. These results indicate that fetal NPC are responsive to cerebral ischaemia factors, and the migratory cue is likely to be a soluble extracellular factor(s). To investigate the factors present in ischaemic tissue was beyond the scope of this study. However we tested factors that had been previously reported to be up-regulated following cerebral ischaemia, and to be associated with enhanced neurogenesis and neuroblast migration in our in vitro migration assays. We found that neurospheres were capable of migration in response to FGF-2 and EGF but did not migrate extensively in the presence of VEGF (data not shown). In contrast to the NPC migratory response observed in vitro, we did not observe any major cell migration from the injection site to the ischaemic striatum or cortex. It is possible that the soluble cues promoted cell migration in vitro because of the absence of inhibitory or toxic molecules present in the in vivo environment. It is also conceivable that the FePro-labelled NPCs were phagocytosed by macrophages in vivo⁴⁵. The apparent lack of accumulation of NPCs at the infarcted site correlates with the lack of a significant reduction in infarct size. These findings are consistent with a recent report showing that reduction in infarct size is “dose-dependent” and that neural stem cell injection is effective in reducing neural damage only following a moderate infarct⁴⁶. On the other hand, behavioural improvement following injection of NPC has been reported in the absence of a significant reduction in infarct size, and might be attributed to increased plasticity in the grafted brains, possibly via paracrine mechanisms as reported for both neural and non-neural stem cells^16,47–49.

In our study, NPCs were engrafted at 48 hours post-ischaemia. Data from the in vitro NPC migration assay demonstrated that NPCs responded to migratory cues from this time point, except at the highest concentration. In a recent study, Darsalia et al. demonstrated that this time point was optimal for cell survival and migration in a similar 30 minute occlusion model and intrastriatal engraftment of human fetal NPCs³. However, engraftment at 48 hours post-ischaemia may not be the optimal time point to expect to have an effect on infarct volume. Therefore, functional assays would be important in future studies to establish a therapeutic effect of cell engraftment and cell labelling with MRI contrast agents. One study reported that engraftment of cells labelled with a gadolinium compound reduced functional recovery compared to engraftment of cells labelled with a fluorescent dye only⁵⁰. Cell migration has been observed in the corpus callosum in other studies engrafting murine embryonic stem cells or human NPC at later timepoints in models of cerebral ischaemia. Injection at the corpus callosum allows for cell injection at a distance from the lesion, reducing exposure to potentially toxic inflammatory environment, while providing a potential pathway for migration towards the lesion site and subsequent migration into the lesion. Clearly, cell type and origin, engraftment timepoint, engraftment site and infarct size are important factors for survival and migration of engrafted cells in ischaemic environments, as well as the parameters used to assess recovery.