Tracking of Mesenchymal Stem Cells with Fluorescence Endomicroscopy Imaging in Radiotherapy-Induced Lung Injury

Jessica R Perez, Norma Ybarra, Frederic Chagnon, Monica Serban, Sangkyu Lee, Jan Seuntjens, Olivier Lesur, Issam El Naqa, Jessica R Perez, Norma Ybarra, Frederic Chagnon, Monica Serban, Sangkyu Lee, Jan Seuntjens, Olivier Lesur, Issam El Naqa

Abstract

Mesenchymal stem cells (MSCs) have potential for reducing inflammation and promoting organ repair. However, limitations in available techniques to track them and assess this potential for lung repair have hindered their applicability. In this work, we proposed, implemented and evaluated the use of fluorescence endomicroscopy as a novel imaging tool to track MSCs in vivo. MSCs were fluorescently labeled and injected into a rat model of radiation-induced lung injury via endotracheal (ET) or intravascular (IV) administration. Our results show that MSCs were visible in the lungs with fluorescence endomicroscopy. Moreover, we developed an automatic cell counting algorithm to quantify the number of detected cells in each condition. We observed a significantly higher number of detected cells in ET injection compared to IV and a slight increase in the mean number of detected cells in irradiated lungs compared to control, although the latter did not reach statistical significance. Fluorescence endomicroscopy imaging is a powerful new minimally invasive and translatable tool that can be used to track and quantify MSCs in the lungs and help assess their potential in organ repair.

Figures

Figure 1. In vivo fluorescence endomicroscopy imaging…
Figure 1. In vivo fluorescence endomicroscopy imaging of labeled MSCs in the lungs for each injection route and condition.
Representative images from video sequences. MSC appear as bright spots (examples of cells shown by red arrows). (a) Ctrl-MSC ET. (b) RT-MSC ET. (c) Ctrl-MSC IV. (d) RT-MSC IV. Cells appear brighter and more numerous in ET injection (top row) compared to IV (bottom row). More cells are observed in irradiated lungs (right) compared to controls (left).
Figure 2. Automatic cell counting algorithm for…
Figure 2. Automatic cell counting algorithm for image quantification.
Each frame in the video sequence is treated as a stand alone image. (a) Summary of workflow. (b) Original representative image. (c) First contrast is enhanced followed by granulometry (d) to determine objects size and image opening. (e) Then, a threshold is applied to highlight bright cells. (f) Finally, cells are counted with connected component analysis.
Figure 3. Automatic cell counting algorithm validation.
Figure 3. Automatic cell counting algorithm validation.
Graph represents number of counted cells for each random frame comparing inter-observer variability and visual vs automatic counting with residual differences. (a) Inter-observer variability ET. (b) Inter-observer variability IV. (c) Visual vs Automatic ET. (d) Visual vs Automatic IV.
Figure 4. Number of detected cells per…
Figure 4. Number of detected cells per video frame in a representative video sequence.
Representative cell count per frame using the automatic cell counting algorithm for ET (a) and IV (b). As the microendoscope probe moves in the lungs acquiring images, the number of detected cells varies.
Figure 5. Mean number of detected cells…
Figure 5. Mean number of detected cells for intravascular and endotracheal injection.
The mean number of detected cells was computed for each video and each rat (each point is one rat with two videos per rat). The horizontal bars represent the mean and standard deviation. The mean number of detected cells differed significantly with 0.5 for IV and 3.6 for ET (p = 0.02).
Figure 6. Mean number of detected cells…
Figure 6. Mean number of detected cells in control and irradiated rats for intravascular and endotracheal injection.
Each point represents a video (two videos per rat) and the horizontal bars represent the mean with 95% CI. A higher mean number of detected cells was observed in the radiation group for both ET with 3.6 for control and 5.4 for irradiated and IV with 0.5 for controls and 0.8 for irradiated. However, those differences were not statistically significant.
Figure 7. Fluorescence microscopy of lung sections…
Figure 7. Fluorescence microscopy of lung sections post-endomicroscopy imaging.
MSCs appear red stained for in vivo imaging with DiD. Nuclei stained blue with DAPI. (a) Lung sections of rat where MSCs were injected ET and (b) IV.

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