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Detection of Iron Oxide Nanoparticles (IONPs)-Labeled Stem Cells Using Quantitative UTE Imaging

Jiyo S Athertya¹, Johnny Akers², Sophia Dwek¹, Zhao Wei¹, Jiang Du¹, Eric Y Chang^1,3, Mya Thu², and Hyungseok Jang¹
¹Radiology, University of California San Diego, San Diego, CA, United States, ²VisiCELL Medical Inc, San Diego, CA, United States, ³Radiology Service, VA San Diego Healthcare System, San Diego, CA, United States

Synopsis

Non-invasive, clinically applicable tracking of therapeutic cells by magnetic resonance imaging (MRI) offers unparalleled insight into the safety and efficacy of cell-based therapies in the body. Here we used a series of 3D quantitative UTE techniques including UTE-QSM, UTE-T₁, and UTE-T₂^* mapping to evaluate the MR characteristics of stem cells labeled with a proprietary nanoparticle formulation that simultaneously labels cells for both optical and MR imaging. In a phantom experiment, all quantitative UTE parameters showed strong correlation with concentrations of labeled stem cells. Interestingly, in ex vivo mouse imaging, only UTE-QSM and UTE-T₂^* mapping detected the injected, labeled stem cells.

Introduction

Stem and immune cell therapy offers promising treatment solutions for many diseases in oncology and regenerative medicine applications, including Parkinson’s and multiple sclerosis¹. Non-invasive imaging of cellular therapeutics in the subjects during and after cell therapy is critical for assessing safety and understanding efficacy for an effective treatment strategy. A variety of iron oxide nanoparticles (IONPs)-based stem cell labeling agents have been utilized for MRI-based cell tracking^2–4, including ferumoxytol, a magnetically active FDA-approved intravenous iron supplement that can be used off-label as an MRI-visible stem cell labeling agent^5,6. However, ferumoxytol’s T₂^* decreases as its concentration increases^5,7, making higher densities of ferumoxytol-labeled stem cells a difficult detection target for clinical MRI. Ultrashort echo time (UTE) sequences are able to detect signals from short T₂^* components such as iron^8–10. In this study, the efficacy of a series of 3D quantitative UTE (qUTE) techniques was investigated for the detection and quantification of IONPs-labeled stem cells.

Methods

Cell labeling: Human neural stem cells (NSCs) were labeled using VMI-Trac Dual, a proprietary nanoparticle formulation that labels cells with ferumoxytol and NIR dye that together enable cell tracking through optical and magnetic resonance imaging (MRI), according to manufacturer’s protocol (Visicell Medical Inc., La Jolla, CA). Treated cells were washed to remove unincorporated nanoparticles, fixed with 4% paraformaldehyde, and examined by Prussian Blue staining and fluorescence microscopy.
Phantom design: Figure 1A shows the phantom that was prepared for validation of qUTE imaging. Specified numbers of unlabeled (2500 cell/mL, D0) and dual-labeled (2500 and 7500 cell/mL, D1 and D2) NSCs were suspended in 0.4% low melt agarose and layered in 3-mL syringes. A syringe of NSCs labeled with ferumoxytol (2500 cell/mL, S1) was included as a positive control (single-labeled).
Ex vivo mouse imaging: 3120, 6250, 12500, 25000, or 50000 labeled NSCs were injected into the mammary fat pads of a 14-week-old NSG™ mouse. 6250 and 12500 labeled NSCs were also injected intramuscularly into the mouse’s gastrocnemius. All injections were 50ml. Post-injection, imaging was performed on an IVIS Spectrum. Post-imaging, the mouse was euthanized and underwent MR imaging.
MR Imaging: Images were acquired using the Carr Purcell Meiboom Gill (CPMG) T₂ mapping sequence and 3D UTE-Cones-based quantitative susceptibility mapping (UTE-QSM)⁸, variable flip angle UTE-T₁ (VFA-UTE-T₁) mapping¹¹ and UTE-T₂^* mapping¹² sequences, as shown in Figures 1B and 1C. The MRI parameters are shown in Figure 1D.
Data Processing: The data analysis algorithm was written in MATLAB. For T₁ and T₂^* calculation, the Levenberg-Marquardt method was used for nonlinear least-squares curve fitting. For the QSM calculation, morphology enabled dipole inversion (MEDI)¹³-based QSM reconstruction was utilized. The T₂ map was calculated using the toolkit on the scanner along with images acquired with the CPMG sequence.

Results

Figure 2A shows that 100% of the labeled NSCs stained positive for Prussian blue and were visualized by fluorescence microscopy, illustrating efficient loading of NIR dye and ferumoxytol into stem cells by VMI-Trac Dual. After injection into the gastrocnemius muscle (Figure 2B) or mammary fat pads (Figure 2C), labeled cells could be tracked by optical imaging on an IVIS scanner. Figure 3 shows susceptibility, UTE-T₁, UTE-T₂^*, and CPMG-T₂ parameter maps of the unlabeled and labeled stem cells in the phantom. The estimated magnetic susceptibility, R₁, R₂^*, and R₂ all showed nearly perfect linear correlations with the density of labeled cells (R² > 0.99). In the ex vivo experiment with an NSC-injected mouse, qUTE techniques detected a total of six injected regions (I₂-I₇) out of seven possible regions. Figures 4 and 5 show all parameter maps in the corresponding slices. UTE-QSM showed elevated susceptibility in the regions injected with the labeled stem cells (I₂-I₇) more clearly than other techniques. UTE-T₂^* mapping exhibited decreased relaxation times in the most injected regions (I₂-I₇) but was obscured by surrounding tissues with high inhomogeneity. UTE-T₁ and CPMG-T₂ showed no significant changes in relaxation times of the injected regions.

Discussion and Conclusion

In this study, we demonstrated that IONPs-labeled stem cells could be detected by qUTE-MR techniques including UTE-QSM, UTE-T₁, and UTE-T₂^* mapping. Although this study’s qUTE and CPMG-T₂ techniques showed feasibility in quantification and localization of IONPs-labeled stem cells in the phantom experiment, only UTE-QSM and UTE-T₂^* demonstrated quantitative changes in the injected regions of the ex vivo mouse, with UTE-QSM showing the best performance. As most soft tissues are slightly diamagnetic in a living system, paramagnetic IONPs with high positive susceptibility can provide high contrast in UTE-QSM. UTE-T₂^* is also an effective approach in this regard as T₂^* depends on susceptibility related dephasing (i.e., T₂’), but because T₂^* only depends on the magnitude of susceptibility, not the positive or negative nature of susceptibility (e.g., bone vs. iron), T₂^* is prone to the inhomogeneity of the surrounding tissues. The shortest T₁ (D2) measured in the phantom was 1.57 seconds. This explains why T₁ was not effective in the ex vivo mouse imaging. Since most tissues showed a T₁ value below 1.5 seconds, the labeled stem cells with long T₁ could not be detected with high contrast. Further studies are planned to investigate these findings in further depth in vivo.

Acknowledgements

The authors acknowledge grant support from the NIH (R01AR062581, R01AR068987, R01AR075825, R01AR078877, and R21AR075851), Veterans Affairs (I01RX002604, I01CX002211, and I01CX001388), and GE Healthcare.

References

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Figures

Figure 1. Experimental setup. (A) NSC phantom design, (B) pulse sequence diagram of dual echo 3D UTE-Cones, (C) 3D spiral cones trajectory, and (D) MR imaging parameters. NSC phantom contains single-labeled NSC as a positive control as well as NSCs with three different iron concentrations. Interleaved dual echo UTE-Cones imaging was performed to acquire data for UTE-QSM and UTE-T₂^*, while single echo imaging was performed for UTE-T₁ mapping, and CPMG imaging was performed for T₂ mapping.

Figure 2. VMI-Trac Dual labels cells for dual modality imaging. (A) Fluorescence microscopy analysis and Prussian Blue staining of labeled NSCs. IVIS imaging of labeled NSC injected into gastrocnemius (B) and mammary fat pads (C) of an NSG™ mouse.

Figure 3. Phantom experiment. All parameters show a high linear relationship with the number of labeled cells (0, 2500, and 7500 cells/µL). The measured susceptibility is 0.03, 0.30, 1.18, and 0.05 ppm for D0, D1, D2, and S1, respectively. The measured T₁ is 3.3, 2.5, 1.6, and 2.8 sec for D0, D1, D2, and S1, respectively. The measured T₂^* is 666.9, 7.5, 2.6, 13.3 msec for D0, D1, D2, and S1, respectively. The measured CPMG-T₂ is 360, 130, 59, 168 msec for D0, D1, D2, and S1, respectively.

Figure 4. Injection points (I₂, I₃, and I₄) and MRI parameters of the NSC-injected mouse. Magnetic susceptibility from UTE-QSM and UTE-T₂^* detected the injected NSC, whereas UTE-T₁and CPMG-T₂ showed no substantial changes in the relaxation times of the NSC-injected regions.

Figure 5. Injection points (I₅, I₆, and I₇) and MRI parameters of the NSC-injected mouse. Magnetic susceptibility from UTE-QSM achieved clear detection of the injected NSCs with strong positive values (i.e., paramagnetic). UTE-T₂^* also showed decreased relaxation times in the NSC-injected region but was slightly obscured by the inhomogeneous T₂^* values of the surrounding tissues.

Proc. Intl. Soc. Mag. Reson. Med. 30 (2022)

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DOI: https://doi.org/10.58530/2022/2457