In vivo two-photon microscopy reveals the contribution of Sox9+ cell to kidney regeneration in a mouse model with extracellular vesicle treatment

Abstract

Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have been shown to stimulate regeneration in the treatment of kidney injury. Renal regeneration is also thought to be stimulated by the activation of Sox9⁺ cells. However, whether and how the activation mechanisms underlying EV treatment and Sox9⁺ cell-dependent regeneration intersect is unclear. We reasoned that a high-resolution imaging platform in living animals could help to untangle this system. To test this idea, we first applied EVs derived from human placenta-derived MSCs (hP-MSCs) to a Sox9-Cre^ERT2; R26^mTmG transgenic mouse model of acute kidney injury (AKI). Then, we developed an abdominal imaging window in the mouse and tracked the Sox9⁺ cells in the inducible Sox9-Cre transgenic mice via <i>in vivo</i> lineage tracing with two-photon intravital microscopy. Our results demonstrated that EVs can travel to the injured kidneys post intravenous injection as visualized by Gaussia luciferase imaging and markedly increase the activation of Sox9⁺ cells. Moreover, the two-photon living imaging of lineage-labeled Sox9⁺ cells showed that the EVs promoted the expansion of Sox9⁺ cells in kidneys post AKI. Histological staining results confirmed that the descendants of Sox9⁺ cells contributed to nephric tubule regeneration which significantly ameliorated the renal function after AKI. In summary, intravital lineage tracing with two-photon microscopy through an embedded abdominal imaging window provides a practical strategy to investigate the beneficial functions and to clarify the mechanisms of regenerative therapies in AKI.