title: Multimodal imaging to evaluate the distribution and fate of a mesenchymal stromal cell therapy
Creators: name: Hernandez Pichardo, Alejandra
Publisher: name: University of Liverpool Repository
Year: 2022

description: Mesenchymal stromal cell (MSC) treatments have shown beneficial outcomes in preclinical models of various diseases, but limited therapeutic effects in clinical trials. This disparity in translation reflects the need to understand the mechanisms involved in the host’s response to therapy. Intravenous injection is the preferred delivery method in the clinics, but it has been observed that using this route leads to MSCs becoming entrapped in the lungs, making this organ an interesting study target. In this thesis, different imaging modalities to study the distribution and fate of administered MSCs in the lung were used. The first aim was to combine the sensitivity of bioluminescence imaging (BLI) with the ability of micro-computed tomography (micro-CT) to image lung tissue to track the cells in vivo. Then, to study the biodistribution of the MSCs in the lung microenvironment at single-cell resolution, an optical tissue clearing protocol was established. Finally, the effect of the MSCs on innate immune cells in the lung and their potential interactions were investigated. Human umbilical cord MSCs (hUC-MSCs) that had been labelled with the genetic reporter Firefly luciferase (FLuc), the fluorescent reporter tandem Tomato (tdTomato), or with gold nanorods were used. The hUC-MSCs were injected into the tail vein of mice, which were then imaged in vivo using BLI and micro-CT. After MSC injection, animals were culled, and the lungs collected and processed for confocal microscopy or flow cytometry. BLI revealed that following intravenous injection, the MSCs localized to the lungs hampering the ability of MSOT to image the MSCs within this organ. Using micro-CT, it was not possible to detect the MSCs, indicating that this method might lack sensitivity to image gold-labelled cells. Next, the CUBIC, a modified stabilized DISCO (s-DISCO) and ethyl cinnamate (ECi) optical tissue clearing protocols were compared to find a suitable method for studying the biodistribution of hUC-MSCs and their interactions with the mouse lung microenvironment. CUBIC was the only method that enabled direct imaging of tdTomato-expressing hUC-MSCs as the other methods quenched the fluorescence of the reporter. Moreover, CUBIC in combination with immunofluorescence allowed the interaction of the hUC-MSCs with cells in the host lung to be investigated. Particularly, it was observed that the hUC-MSCs appeared to be retained in the pulmonary microvasculature as they were not found in large blood vessels. Flow cytometric analysis showed that shortly after hUC-MSC IV injection, neutrophils, monocytes, and macrophages mobilized to the lung and participated in an inflammatory response. Twenty-four hours post cell infusion, the number of innate immune cells in the lungs decreased but a polarization toward an anti-inflammatory phenotype was observed. Moreover, immunofluorescent staining revealed that neutrophils were preferentially distributed in close vicinity to the hUC-MSCs, suggesting that their clearance within 24 h might involve efferocytosis. In summary, using a range of in vivo and ex vivo imaging techniques, it was shown that following intravenous injection into mice, hUC-MSCs appeared to accumulate in the pulmonary vessels and mostly died within 24 h. Within 2 h following administration, the hUC-MSCs caused an inflammatory response in the lung, leading to an increase in neutrophils and pro-inflammatory macrophages. However, by 24 h, neutrophils were at basal levels and there was an increase in anti-inflammatory macrophages. Although there are numerous reports indicating that MSCs polarise macrophage towards an anti-inflammatory phenotype, an interesting finding of this study was that the initial effect of hUC-MSCs on host immune cells was actually pro-inflammatory. This may provide some insight into the potential therapeutic mechanisms of MSCs.