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-CreERT2; R26mTmG 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 in vivo 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.