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S1 Fig. Isoflurane becomes concentrated within fat pads during imaging[ 31 ] and these high levels of 19 F signal can lead to false-positive detection and incorrect quantification. Isoflurane anesthesia is preferred for the ease of use and the relatively long imaging times required for 19 F MRI.

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This study demonstrates the ability to use 19 F-MRI cell tracking to detect and, most importantly, to measure the number of transplanted stem cells in vivo. Cell number cannot be quantified using other MRI cell tracking techniques. Numerous studies have reported on the challenges associated with quantification of signal loss due to iron-labeled cells.

In this study we show excellent correlation between the number of labeled cells implanted and the number of cells counted on day 0 by 19 F-MRI. This capability will pave the way for MRI to be used for to confirm the delivery of therapeutic cell transplants. The importance of accurate delivery of cells to a target tissue cannot be overstated. In preclinical investigations often stem cells will be transplanted then a set time allowed to lapse before the transplanted tissue is removed and processed for microscopy, to determine whether stem cells remain in the tissue.

In many cases only a very small sample of the transplanted tissue is evaluated. The histological assessment in this study involved the analysis of 1cm of cord tissue on either side of the transplant site, six weeks after transplantation.

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Without MRI, a negative observation of MSC by microscopy would have been taken as failure to engraft rather than due to a missed injection. Injection confirmation with 19 F MRI would have the additional advantage of determining how many cells were properly injected and remain at the site. In a first-in-man study, MRI and scintigraphy were used to assess the success of intranodal injections consisting of a mixture of iron- or indium-labeled dendritic cell DC for cancer therapy in melanoma patients.

For DC therapy knowledge of the number of cells delivered to a lymph node is especially critical since DC migration to nodes correlates with effective stimulation of T cells. We observed some variation in the number of 19 F atoms loaded per cell between experiments, although the average loading was not significantly different for mouse versus human MSC.

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  4. The cell size is one of the more important factors influencing the labeling with Cell Sense and mouse and human MSC are approximately 30 microns in diameter. The fact that it is possible to obtain robust measurements of the number of cells detected by MRI, despite inter-experiment variation in the number of 19 F atoms per cell, is another positive feature of this type of cell tracking. This is particularly important when considering clinical translation, since 19 F uptake is expected to vary between cell donors. This study also revealed some interesting information about tracking cell fate over time with 19 F MRI.

    The MSC underwent clearance from the sites of implantation in muscle at different rates, which is not unexpected since the two different mouse strains have very different immune systems. The decline in the signal is likely the result of a number of different things happening at the transplant site. First, a large number of MSC will die early after their direct transplantation into a tissue.

    Third, MSC may have migrated away from the implant site; although we did not detect 19 F signal in other nearby locations. In the immune compromised model the 19 F signal persisted in all mice until the experimental endpoint. This would have allowed us to say with more confidence that the 19 F label was associated with macrophages and not the implanted hMSC. Clearance of label and macrophages may have been slower in these mice because of the inhibited immune system and lack of rejection response. Our observation of bystander cell uptake of 19 F cell label is supported by a study by Boehm-Sturm et al.

    The signal from 19 F labeled stem cells persisted for more than 4 weeks after implantation while, over the same time period, the bioluminescence declined, indicating stem cell death. Terrovitis et al. In both cases MRI signal loss due to iron was detected for 3 weeks post cell injection and correlated with the presence of iron containing macrophages in histology. Although the area of signal void decreased over time substantial signal void persisted at the injection site in all mice.

    Since proton MRI is sensitive to even small numbers of iron-labeled cells this form of cell tracking is most susceptible to the misinterpretation of cell fate. Previous studies performed in our lab using iron labeled syngeneic MSC also revealed the persistence of an iron signal void past 21 days in immune competent mice. In summary, 19 F MRI can be used to provide immediate assessment of implanted cells with excellent correlation between implanted cell number and in vivo quantification.

    Over time, as the cells are cleared from the transplantation site, transfer of the 19 F label to bystander cells may confuse interpretation of the change in 19 F signal. With the first-in-man studies of 19 F MRI recently completed, this result will be particularly relevant when translating this technique into the clinic. A Strong isoflurane signal red arrow is detectable following accumulation in the fat pads of mice after excitation with the standard Gaussian filtered sinc pulse.

    This signal is affected by a chemical shift from the fat pad blue arrow , the fluorine signal in the reference tube experiences does not shift. By applying a Gaussian filter to the sinc a more rectangular waveform is achieved after Fourier transform, but at the cost of broadening the pulse in frequency space. C The mouse was then scanned with a non-filtered sinc pulse. Fourier transform of this pulse produces a narrower excitation that did not excite isoflurane 19 F atoms, preventing background signal.

    Both images have been windowed to the same level, and brightened to show the noise distribution. B The filtered pulse shape in time space is shown, with a width of 0. This produces a narrower pulse in frequency space, preventing the excitation of isoflurane signal.

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    Analyzed the data: JG ER. Designed hardware RF coil for data acquisition: KG. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Methods We investigated two common stem cell transplant mouse models: an immune competent, syngeneic transplant model and an immune compromised, xenograft transplant model. Results Immediately following transplantation, 19 F-MRI quantification correlated very well with the expected cell number in both models.

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    Conclusions Our results show that 19 F-MRI is an excellent tool for verifying the delivery of therapeutic cells early after transplantation. Introduction Stem cell therapy has the potential to play an important role in regenerative medicine. MSC implantation 1. MRI All images were collected using a 9. Image analysis and quantification Prior to image analysis, a signal correction was applied to the 19 F datasets by subtracting a constant value x from all voxels within the dataset using the image program, ImageJ. Download: PPT. Fig 1. Cellular viability and loading with the 19 F-agent.

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    Fig 2. In vitro validation of 19 F-MRI quantification accuracy. Fig 3. Comparison of 19 F-labeled cell detection in two transplantation models over time. Fig 4. Representative Day 0 MRI, fluorescence microscopy, and histology acquired as 10x magnification from both implant models. Fig 5.

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    Representative endpoint MRI, fluorescence microscopy, and immunohistochemistry acquired at 10x magnification from both implant models. As I wrote five years ago , those who watch us can expect to be — and indeed are being — increasingly closely watched themselves as the lens gets turned on them:. With attention to detail, good connections in all senses and the application of digital forensics all sorts of discrete data dots can be linked — enabling official narratives to be interrogated and unpicked with technology-fuelled speed.

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