New paradigms in cellular therapies aim to employ reserves of endogenous cell populations (Leker and McKay, 2004
). While the use of MRI for tracking migration of transplanted cells is well established, methodologies for monitoring and quantifying endogenous cell migration are only recently emerging. In this study we have tuned an in vivo
cellular labeling protocol to enable visualization of early migration of NPCs into the OB, facilitating quantification of this phenomenon. During the first week following labeling, a linear accumulation of newly arriving cells in the OB was measured. Initially, at day 1, cells were confined to the RMS; the contrast in the image was spatially compact and reached the entrance to the OB. This resembles the migratory pattern of a tight chain structure that characterizes neuroblasts migration, and is referred to as tangential chain migration (Lois et al., 1996
; Rousselot et al., 1995
; Whitman et al., 2009
). By day 3, cells are observed entering the central portion of the OB; the granule cell layer, where most cells are destined to reside. At day 8, many cells can be detected not only in the central portion of the OB, but also in the outer edges, where some cells can migrate to (Luskin, 1993
). The volume fraction of dark spots in the OB was well correlated with pure ICP-MS determination of iron content, emphasizing the reliability of using the MRI and BioImagesuite software to measure migration of MPIO labeled progenitor cells. This migration rate matches nearly identically to classical neurogenesis studies employing immunohistochemical means, which monitored 70 - 80 μm/h (Luskin, 1993
; Nam et al., 2007
). MRI detection of migration plateaus from week 1 to 2, indicating that the MPIO injection acts like a bolus, not a sustained slow release label. Recently, Sumner et al. published that only 10 - 30 % of the purified cells in the SVZ/RMS region harbored the MPIOs, of these approximately 50 % were found to be GFAP and vimentin positive astrocytes (Sumner et al., 2009
). Thus, the total percentage of labeled neural stem cells can be estimated to range between 5 – 17 %.
Super paramagnetic iron oxide particles are extensively used in cellular MRI for in vivo
cell tracking and several methodologies have been developed for quantification of transplanted labeled cells, including T2
* relaxometry (Dahnke and Schaeffter, 2005
; Politi et al., 2007
), or pixel intensity histograms. Other non direct methods such as flow cytometry (Williams et al., 2007
) or atomic absorption spectrometry (Raschzok et al., 2009
) can only be performed on ex vivo
samples. Phase map cross correlation is a new post processing data analysis technique that was recently developed to quantify localization and accumulation of SPIO particles (Mills et al., 2008
). This method was successfully applied on phantom agar gels with implanted SPIO labeled macrophages, and awaits in vivo
The quantification strategy employed here uses a different paradigm, more akin to spot-detection. Due to the T2
* blooming artifact generated by the MPIOs, labeled cells can be detected as dark contrast at resolutions larger than their physical size (Shapiro et al., 2004
). Thus, identification of dark spots is indicative of a pixel containing labeled cells. Prior calibration of the contrast characteristics from labeled cells aids in thresholding. For example, a 30% drop in signal intensity has been measured for single cells harboring 1.63 micron MPIOs imaged at resolutions used in this study (Shapiro et al., 2005
). Therefore, any pixel which is more than 30% lower signal intensity than the local background was counted in this study as containing labeled cells. Indeed, voxels were detected with contrast changes of less than 30%, but they were not counted as having labeled cells, even though they may well have. In that regard, the analysis used herein was very conservative and some labeled cells likely were not included in the enumerations, however the exact number of cells is not possible to compute currently.
There are still some challenges using spot detection for quantification of iron oxide labeled cells. One fundamental hindrance is that signal voids generated in the gradient echo image could result from intrinsic anatomical properties and not necessarily relate to labeled cells. Furthermore, since the generated susceptibility effect is independent on the cell type, no method yet exists to discriminate the signal arising from different cell types. Thus, it cannot be determined whether the label resides in the original cell population or arises from a different cell through secondary transfer of the label. In addition, we still cannot resolve the differences between signal voids produced by extracellular or intracellular particles. Considering these limitations, we applied several constrains to our quantification approach. First, we quantified the early migratory rates during the initial 2 weeks post injection. During this restricted time frame we assume low percentage of apoptosis of migrating cells, as it was reported that the high mortality rate occurs 15 to 45 days after the cells were born (Petreanu and Alvarez-Buylla, 2002
; Winner et al., 2002
). According to Sumner et al, 4%–13% of the labeled cells in the OB are microglia (Sumner et al., 2009
), thus the prominent population of labeled cells in the OB lineage is from migrating progenitor cells. Second, in order to avoid false positive selection of anatomical structures, every dark pixel was manually investigated on three orthogonal MRI views. Thus, we have used semi automated quantification and have chosen only those dark pixels that could arise from migrating cells. In addition we have avoided selection of dark pixels that are located in the proximity of the edge of the brain, an area in which by fluorescence microscopy imaging (unpublished data), we often observe fluorescent particles that non-specifically attached to the rim of the brain.
The challenge to achieve accurate numbers of migrating cells following endogenous labeling of precursor cells is still in its infancy. The in vivo
labeling efficiency has yet to be determined and amounts of iron to cell numbers have yet to be correlated. For that goal, Magnetic Particle Imaging (MPI) could potentially be applied to correlate magnetic field maps to amount of iron and presumably cell numbers (Weizenecker et al., 2009
). Critical for this might be methods for modulating intracellular versus extracellular differences in relaxivity of the particles, thus determining whether the signal arises from labeled cells. Work in this direction is currently underway.