Introduction

Stem cells are a potentially useful treatment for chronic lung diseases, such as asthma, chronic obstructive pulmonary disease (COPD) and bronchopulmonary dysplasia (BPD).¹ It has been shown that the major innate immune cells in the lungs, alveolar macrophages, can be derived from pluripotent embryonic stem cells and these alveolar-like macrophages (ALMs) promote repair of lung disease in animal models.² Translation of this approach to the clinic will benefit from imaging methods that can detect and monitor ALMs in vivo following instillation in the lungs. It has been demonstrated that superparamagnetic iron oxide nanoparticles (SPIONs) can enable proton MRI of cells in the lung.³ Hyperpolarized (HP) MRI provides a further improvement in detection sensitivity of SPION-labeled cells in the lung.⁴ In this work, we demonstrate the use of HP ¹²⁹Xe MRI combined with SPION labeling for detection of ALMs in the rat lung.

Methods

ALMs were produced following the method of Litvack et al² and loaded with varying concentrations of green fluorescent-labeled SPIONs (Molday ION EverGreen™). After four hours, confocal fluorescence microscopy and flow cytometry were used to confirm SPION uptake by the cells. The effects of SPIONs on cell viability after incubation with 0, 0.5, 1, 2, and 4% SPIONs (v/v) for four hours was measured using a Presto Blue® assay. As a preliminary step in the detection of ALMs in the lung, the effects of localized instillation of (i) phosphate buffer saline (PBS), (ii) PBS + SPIONs, and (iii) SPION-labeled ALMs on HP ¹²⁹Xe signal acquired from the lungs of separate healthy Sprague Dawley rats in vivo were investigated. ALMs were incubated with a 4% SPION solution (v/v) for four hours and approximately two million cells were resuspended in 100 µL of PBS. Imaging was performed in anesthetized mechanically ventilated rats during a ten second breath-hold using a 3D gradient-recalled echo (GRE) sequence (FOV = 70 × 70 mm, matrix = 64 × 64, TR = 17 ms, TE = 3.13 ms, flip angle = 2°). Solutions of 100 µL of PBS, PBS + SPIONs and ALMs were delivered via a tracheostomy to either the right or left lung using a 24-gauge catheter. Imaging was performed approximately five minutes after instillation of all solutions and one hour later. Signal-to-noise ratios (SNR) were calculated before and as a function of time after instillation and normalized to the SNR of the main airways.

Results

The confocal microscopy image in Fig. 1(a) shows the ALM nuclei (blue), confirming that SPIONs (green) have been sequestered. The fluorescence peak shift in Fig. 1(b) indicates that larger quantities of SPIONs are sequestered as the SPION concentration increases. Figure 2 summarizes the effects of SPIONs on cell viability in vitro indicating about 80% of cells survive after four hours of co-incubation with SPIONs. Figure 3 shows representative coronal ¹²⁹Xe images before and following instillation of (a) PBS, (b) PBS + SPIONs, and (c) labeled ALMs. Localized ¹²⁹Xe image signal hypointensities (shown by red circles) were observed five minutes following all instillations. Hypointensities resolved one hour following instillation of PBS and PBS + SPIONs (Fig. 3(a) and (b)), but were still observable one hour following instillation of the SPION-labeled cells (Fig. 3(c)). Figure 4 shows the normalized ¹²⁹Xe SNR values as a function of time calculated from the regions of interest shown in Fig. 3, demonstrating the persistence of signal drop for the SPION-labeled ALMs.

Discussion

Cell assays demonstrate that ALMs readily take up SPIONs and remain at least 80% viable after four hours of incubation with varying SPION concentrations. After instillation of SPION-labeled ALMSs, the lungs appear to retain hypointensities associated with the SPION-labeled ALMs for at least one hour following instillation, unlike the signal hypointensities observed after instillation of PBS and PBS + SPIONs. This is likely due to retention of the SPION-labeled ALMs and clearance of the PBS/SPIONs from the lungs by ventilation. These preliminary results suggest that this method could potentially be used to detect and monitor ALMs in the lungs in vivo, regionally and longitudinally. In the future, the locations of the cells detected with HP ¹²⁹Xe MRI will be confirmed by fluorescence microscopy. The next steps include prolonged (hours/days) longitudinal imaging of animals before and after instillation. For detection at longer time points, ALMs can be instilled a few days before imaging to allow the cells to adhere to the lung tissue. This proof-of-concept study will serve as a basis for developing stem cell treatment of lung diseases in rat models in the future.

Acknowledgements

The authors would like to thank the Ontario Institute for Regenerative Medicine and Medicine by Design for the New Ideas grant funding this project. VR is funded by a Natural Science and Engineering Research Council of Canada (NSERC) CGS Master’s scholarship and a Restracomp scholarship from the Hospital for Sick Children. Special thanks to members of the Santyr lab for their help with imaging and to the Post lab members for their help with cell work.