Non-invasive real-time imaging modalities, such as MRI, will play a critical role in the clinical application of stem cell therapy across the spectrum of human age and disease, particularly in the CNS which is otherwise inaccessible to visual inspection 27, 28
. Such monitoring likely will be mandatory for clinical trials that precede acceptance for routine therapy, given that such trials will focus on insuring long-term safety of cell-based interventions. MRI provides the ability to assure targeted implantation, track migration and cell-host interactions, validate and quantify reparative responses, monitor cell proliferation, and assess any adverse impacts on host parenchyma and vasculature. The latter two functions may be critical for knowing when to trigger a suicide gene within the transplanted NSCs, a likely requirement by most regulatory agencies. Our current study provides a framework for such translational research, using as proof-of-principle, NSCs in neonatal HII, a plausible early target for stem cell-based therapies. Our study was not designed to investigate the therapeutic effectiveness of NSCs but rather to evaluate whether NSCs rendered “visible” to MRI with iron-labeling could be effectively and safely visualized and monitored for extended time periods without adversely altering either stem cell behavior, status of the host to support engraftment, or the baseline disease condition. Furthermore, the success of murine NSCs to remain viable within the rat brain is well known 29, 30
Animals subjected to HII and followed long-term (for 58 weeks) did evince significant behavioral deficits due to ongoing cavitation of the lesion (), but this effect was not worsened by NSCs and NSCs implanted into uninjured animals did not create behavioral, functional, or histological problems. The purpose of the study was one of visualization of NSCs and not optimizing NSC dose, time of administration, location of administration(s), number of cotemporaneous or sequential administrations, adjunctive agents, or accompanying motor and cognitive enrichment for optimal therapeutic benefit. Indeed, the NSCs were not implanted into a region that would likely have yielded optimal benefit – directly in the penumbra (by virtue, in part, of their known anti-inflammatory, anti-cavitation actions). Rather, the NSCs were administered distant from the lesion to be able to determine whether MRI could track and quantify the dynamic of their migration and proliferation. However, with the knowledge that MRI can be used in this regard and that iron-labeling of NSCs is safe and will not alter the profile of the disease itself, in future studies parameters for maximizing efficacy and benefit can now be systematically tested – in a non-invasive manner in real-time for a given subject within a large cohort of animals where intermittent or repeated treatment in multiple locations might be feasible.
Long-term detection of viable mNSCs by MRI
This is the first report to document the ability of MRI to monitor iron-labeled NSCs in a model of neonatal HII for up to 58 weeks, essentially the lifespan of a laboratory rat. While it is known that NSCs can be visualized using MRI as they migrate to the injury site 5, 7, 25, 31
, the majority of studies have been of relatively short duration 32 33–35 36 37
. The one long-term follow-up study 38
did not involve the developing neonatal
brain which is an entirely different CNS “terrain”. Indeed, a number of adult studies suggest that implanted stem cells may not
retain their iron label 39
. While more study is required to harmonize the results from disparate studies, differences in iron-labeled compounds, injury models, imaging methods, time points and histological endpoints combined with age related changes likely account for the variability with regard to long-term visualization of NSC within the brain. We describe a method by which NSCs (based on signal voids on T2 imaging) could be monitored accurately throughout the 58 week period as confirmed by histology. Indeed, the MRI resolution may have been good enough to detect small clonal
populations of NSCs ().
MR susceptibility studies have suggested that MRI may not only overestimate the hypointensity on T2 or other imaging modalities 40
but also the number of iron-labeled NSCs seen on MR could be an over-representation of the actual number of NSCs present 9, 41
. In our study we observed greater MRI hypointensities than that seen with Prussian Blue staining and future histological studies will need to be correlated to the loss of MR signal. The return of NSC volume to control levels could be the result of numerous processes, including: (i) loss of detection limits by MRI, (ii) migration of NSC-derived cells away from the region of initial integration, and (iii) discharge (and hence loss) of iron particles during proliferation and/or cell death.. While other potential explanations remain plausible, the most likely explanation is that the system prunes cells over time that are no longer necessary for the repair process and/or have not made synaptic connections or have simply become senescent. Stereological criteria were utilized to determine cellular densities from MR-based images, similar to those from histological results 42, 43
Monitoring migration over long time periods
Using MRI, it was possible to calculate the speed at which these NSCs migrate
in the injured neonatal brain 6, 44–46
. Migration occurred within 4–10 days at ~100–125 μm/day. This rate is almost double that seen in adult brains 47–49
, information that is important for planning newborn stem cell-based trials. Given that it is widely acknowledged that a relatively narrow “window-of-opportunity” for neuroprotection or neural repair exists after ischemic injury – typically within the first 10 days – it is significant that potentially reparative NSCs might engage the injury site more quickly and in greater numbers within that “window” in the developing than in the adult brain. Water content is higher and tissue density/compactness lower in the developing mammalian brain and may provide less of a structural barrier to cell movement within parenchymal tissues 50, 51
. Knowledge of such a tempo may provide guidance as to how often and when to monitor for adverse events.
Monitoring proliferation over long time periods
One of our more useful (and novel) findings is that the volume of T2 hypo-intensities could provide a real-time non-invasive
surrogate measure for cell proliferation by calculating a “proliferation index” (CPI). These inferences were entirely consistent with the known dynamics of BrdU incorporation using the same mNSCs implanted in the same manner in the same newborn mouse RVM HII model 7
. Such monitoring will be critical during any clinical use of NSCs.
NSC viability and location
Another important consideration is the early loss of a portion of NSCs after implantation. Several studies have suggested that up to 50% of implanted cells may be lost 11, 52, 53
via toxic inflammatory molecules, free radicals, excess intracellular calcium and excess extracellular glutamate in response to the initial injury 54
. However, when implanted during the proper post-injury interval, NSCs appear to survive and flourish. Our data suggest that MRI might be used to ascertain the appropriate time points for implantation following injury.