Human mesenchymal stem cells (hMSCs) have gained intense research interest due to their immune-modulatory, tissue differentiating, and homing properties to sites of inflammation. Despite evidence demonstrating the biodistribution of infused hMSCs in target organs using static fluorescence imaging or whole-body imaging techniques, surprisingly little is known about how hMSCs behave dynamically within host tissues on a single-cell level in vivo. Here, we infused fluorescently labeled clinical-grade hMSCs into immune-competent mice in which neutrophils and monocytes express a second fluorescent marker under the lysozyme M (LysM) promoter. Using intravital two-photon microscopy (TPM), we were able for the first time to capture dynamic interactions between hMSCs and LysM+ granulocytes in the calvarium bone marrow of recipient mice during systemic LPS challenge in real time. Interestingly, many of the infused hMSCs remained intact despite repeated cellular contacts with host neutrophils. However, we were able to observe the destruction and subsequent phagocytosis of some hMSCs by surrounding granulocytes. Thus, our imaging platform provides opportunities to gain insight into the biology and therapeutic mechanisms of hMSCs in vivo at a single-cell level within live hosts. 1. Introduction Human mesenchymal stem cells (hMSCs) are self-renewing precursor cells capable of differentiating into bone, adipose tissue, cartilage, and stromal cells of the bone marrow depending on the stimuli [1]. Available data indicate that hMSCs are pericytes whose pleiotropic nature allows them to sense and respond to inflammatory processes in the microenvironment [2]. Although hMSCs are found at low frequency in vivo in a variety of adult tissues including bone marrow, muscle, fat, and dermis, they can be expanded to large numbers under appropriate culture conditions. For this reason, hMSCs have been applied therapeutically in rapidly expanding clinical investigations including more than 200 currently active clinical trials worldwide [1, 3–6]. A wide range of diseases including diabetes, atherosclerosis, multiple sclerosis, systemic lupus, Crohn’s disease, myocardial infarction, stroke, Parkinson’s disease, bone and cartilage repair, wound healing, and graft-versus-host disease [1, 3] have been treated using hMSCs as a cellular therapy. These clinical trials aimed to explore the therapeutic potential of hMSCs with regard to their immune-modulatory properties, tissue regenerative capacity, graft enhancement, tissue protection, and repair capabilities. Similarly, hMSCs have been
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