Effects of the iron oxide nanoparticle Molday ION Rhodamine B on the viability and regenerative function of neural stem cells: relevance to clinical translation

Abstract

An essential component of developing successful neural stem cell (NSC)-based therapies involves the establishment of methodologies to noninvasively monitor grafted NSCs within brain tissues in real time. In this context, ex vivo labeling with ultrasmall superparamagnetic iron oxide (USPIO) particles has been shown to enable efficient tracking of transplanted NSCs via magnetic resonance imaging (MRI). However, whether and how USPIO labeling affects the intrinsic biology of NSCs is not thoroughly understood, and remains an active area of investigation. Here, we perform a comprehensive examination of rat NSC survival and regenerative function upon labeling with the USPIO, Molday ION Rhodamine B (MIRB), which allows for dual magnetic resonance and optical imaging. After optimization of labeling efficiency, two specific doses of MIRB (20 and 50 μg/mL) were chosen and were followed for the rest of the study. We observed that both MIRB doses supported the robust detection of NSCs, over an extended period of time in vitro and in vivo after transplantation into the striata of host rats, using MRI and post hoc fluorescence imaging. Both in culture and after neural transplantation, the higher 50 μg/mL MIRB dose significantly reduced the survival, proliferation, and differentiation rate of the NSCs. Interestingly, although the lower 20 μg/mL MIRB labeling did not produce overtly negative effects, it increased the proliferation and glial differentiation of the NSCs. Additionally, application of this dose also changed the morphological characteristics of neurons and glia produced after NSC differentiation. Importantly, the transplantation of NSCs labeled with either of the two MIRB doses upregulated the immune response in recipient animals. In particular, in animals receiving the 50 μg/mL MIRB-labeled NSCs, this immune response consisted of an increased number of CD68⁺-activated microglia, which appeared to have phagocytosed MIRB particles and cells contributing to an exaggerated MRI signal dropout in the animals. Overall, these results indicate that although USPIO particles, such as MIRB, may have advantageous labeling and magnetic resonance-sensitive features for NSC tracking, a further examination of their effects might be necessary before they can be used in clinical scenarios of cell-based transplantation.

Keywords:

• MRI
• neural stem cells
• iron oxide nanoparticles
• USPIO

Supplementary materials

Figure S1 Optimization of MIRB dose.

Notes: Based on studies regarding MIRB labeling of NSCs reported in literature, NSCs were treated first with 5, 10, 20, 50, 60, 80, or 100 μg Fe/mL of MIRB for a time period of 30 or 48 hours (A–D). Here, an obvious reduction in cell viability was noted even at the 5 μg dose (A and C). Therefore, we reduced the labeling time to 18 hours, again after reviewing the literature. Under these conditions, cells labeled with 5, 10, 20, 50 μg doses did not qualitatively show a significant reduction in cell viability (E–H and J–M). As shown, a loss of cells was already seen at the 60 μg level (I and N). The 20, 50, and 60 μg doses showed good labeling, as confirmed via the Prussian blue staining (G–I) and the detection of rhodamine autofluorescence (L–N). We further increased the incubation period to 20 hours to refine the labeling in cells treated with 20 and 50 μg/mL MIRB (O and P). The labeling efficiency (percentage of labeled cells) as well as intensity (quantitation of rhodamine fluorescence), after 20 hours of MIRB labeling, in doses up to 50 μg, is shown in (Q and R). *P<0.05 compared to 10 μg, ^#P<0.05 compared to 20 μg, **P<0.001 compared to 10 μg, one-way ANOVA. Scale bars: 20 μm.

Abbreviations: MIRB, Molday ION Rhodamine B; NSCs, neural stem cells; ANOVA, analysis of variance.

Figure S2 MRI of sham animals.

Notes: Animals injected with buffer, as opposed to cells, were also scanned using 3D gradient-echo MRI. The imaging protocol and parameters were identical to those mentioned in the main paper. As reported in the literature, at all the three time points (3, 7, and 28 days post-transplantation) assessed, signal dropout was seen corresponding to the needle track area (arrows in [A–C]).

Abbreviation: MRI, magnetic resonance imaging.

Acknowledgments

We thank Kayla Yu and Quentin Remley for their technical assistance on the histological aspects of the project. We are also grateful to Doug Cromey (Arizona Cancer Center Imaging Core Cancer Center Support Grant [P30 CA023074]) for his technical support with confocal microscopy and image analysis. This work was supported by intramural funds from The University of Arizona to LM.

Author contributions

AU, SR, and MJC contributed to experimental design, collection and assembly of data, data analysis and interpretation, and manuscript writing. MV contributed to experimental design, collection and assembly of data, data analysis and interpretation, and manuscript revision. EY contributed to collection and assembly of data, data analysis and interpretation, and manuscript revision. TT contributed to conception and design, collection and assembly of data, data analysis and interpretation, and manuscript revision. LM contributed to conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing and revision, financial support, and final approval of manuscript.

Disclosure

The authors report no conflicts of interest in this work.