Intravital imaging and manipulation of the bone marrow stromal network

Intercellular communication through calcium signaling helps to orchestrate cell function across space and time. Bone marrow stromal cells (BMSCs) form a pervasive 3D network to regulate hematopoiesis and immunity via cytokine secretion. To visualize and manipulate this network activity, we developed an intravital calcium imaging and analysis pipeline, followed by single-cell photochemical ablation to disrupt the network. Using the genetically encoded calcium indicator GCaMP6f, the pipeline enabled robust detection of calcium transients, spatiotemporal dynamics, and network connectivity using graph theory. Our results showed that Lipopolysaccharide (LPS)-induced systemic inflammation increased the frequency and speed of intercellular calcium waves (ICWs). Network analysis further revealed enhanced signal correlation initiated by a subset of BMSCs, presumably the “hub” cells. Deactivating the hub cells using targeted photochemical ablation abolished ICWs under LPS challenge. Overall, our calcium imaging and single cell manipulation toolbox offers a new way of studying stromal network function in vivo.