title: INVESTIGATING THE FATE AND IMMUNOLOGY OF INTRAVENOUSLY INJECTED MESENCHYMAL STROMAL CELLS IN MICE
Creators: name: Amadeo, Francesco
Publisher: name: University of Liverpool Repository
Year: 2022

description: Introduction: Mesenchymal stromal cells (MSCs) are multipotent cells showing great promise in pre-clinical studies and currently used in many clinical trials. The regenerative potential of MSCs is mediated, at least in part, by direct and indirect immunomodulatory mechanisms through the secretion of paracrine molecules. However, the mechanism of action behind the beneficial effect of these cells is not fully understood yet, and there are still concerns about possible undesired negative effects associated with the administration of living cells. Bioluminescence imaging (BLI) is widely used in preclinical models to investigate the biodistribution and safety of injected MSCs by monitoring the fate of luciferase-expressing cells following administration. Aims: The present study was performed to (i) compare the short term biodistribution of umbilical cord (UC), bone marrow (BM) and adipose (A) MSCs, (ii) assess the long-term fate and safety of UC MSCs in immunocompromised animals and (iii) investigate the mobilisation of different immune cells following the in vivo administration of UC MSCs. Methods: To generate luciferase-expressing MSCs so that the cells could be visualised in vivo, a lentiviral transduction protocol was optimised by testing different polycations, and then used to investigate the use of two possible reporter genes, Firefly Luciferase (FLuc) or a novel luciferase called AkaLuc. An IVIS Spectrum system was used to characterise the two BLI systems in vitro and to assess their sensitivity in vivo, via the imaging of FLuc and AkaLuc UC-MSCs intravenously (IV) administrated to C57BL/6J albino mice. Because the FLuc was selected as the optimal reporter gene, lentiviral vectors were used to generate FLuc-expressing UC, BM and A-MSCs. The in vivo short term biodistribution was assessed via the imaging of C57BL/6J albino mice up to 7 days post IV administration of FLuc+ MSCs. Severe combined immunodeficient (SCID) and non-obese diabetic (NOD)/SCID mice were used to evaluate the long-term safety of FLuc+ UC MSCs up to 31 days from their administration. Flow cytometry was used to compare the percentage of innate and adaptive immune cells in the blood, the lungs, the bone marrow, and the spleen of C57BL/6 albino mice 2h and 24h following the administration of UC-MSCs. A multiplex analysis performed on the plasma of the animals was used to evaluate the levels of different cytokines, chemokines, soluble receptors, and growth factors directly associated with the immune system. Results: The in vivo comparison of FLuc and AkaLuc showed that the signal intensity of cells expressing AkaLuc was not stronger than FLuc when standard substrate doses were used (30 mM Akalumine-HCl and 47 mM D-Luciferin). By increasing the D-luciferin dose to 144.5 mM, it was possible to increase the light output obtained with the FLuc system, which was then 4.5-fold stronger than AkaLuc. The AkaLuc system was associated with scar formation under the skin of the animals at the site of subcutaneous injection of the substrate, AkaLumine-HCl. For these reasons FLuc was selected as the optimal reporter for subsequent experiments. FLuc+ UC, BM and A-MSCs were entrapped in the lungs of C57BL/6 albino mice after IV administration. Most of the cells (&gt;90% of the UC, ≥94% of the BM and ≥85% of the A) died in the first 24h post administration and almost no signal was detected by day 3. UC-MSCs administered to immunocompromised mice were also entrapped in the lungs and showed a reduction in the signal in the first 24h similar to that observed in immunocompetent animals. Around 25% of the SCID and 45% of the NOD/SCID animals displayed detectable signal up to day 14. One NOD/SCID mouse showed weak signal up to day 31. Following the administration of UC MSCs in C57BL/6 albino mice, flow cytometry analysis revealed an increase in the percentage of neutrophils in the lungs, the blood and the spleen 2h after the administration of the cells. Neutrophil chemoattractants (CCL2, CCL7, Gro-α and IP-10) were also upregulated in the plasma of the animals 2h after the administration of the MSCs. Conclusions: The characterisation of the biodistribution of the cells and the involvement of the immune system show that although MSCs are short-lived in mice they still result in an immunological response that might contribute to a therapeutic effect.