Stem cells are characterized by the ability to renew themselves and to differentiate into specialized cell types, while stem cell therapy is believed to treat a number of different human diseases through either cell regeneration or paracrine effects. Herein, an in vivo and ex vivo near infrared time domain (NIR TD) optical imaging study was undertaken to evaluate the migratory ability of murine adipose tissue-derived multipotent adult stem cells [mAT-MASC] after intramuscular injection in mice. In vivo NIR TD optical imaging data analysis showed a migration of DiD-labelled mAT-MASC in the leg opposite the injection site, which was confirmed by a fibered confocal microendoscopy system. Ex vivo NIR TD optical imaging results showed a systemic distribution of labelled cells. Considering a potential microenvironmental contamination, a cross-validation study by multimodality approaches was followed: mAT-MASC were isolated from male mice expressing constitutively eGFP, which was detectable using techniques of immunofluorescence and qPCR. Y-chromosome positive cells, injected into wild-type female recipients, were detected by FISH. Cross-validation confirmed the data obtained by in vivo/ex vivo TD optical imaging analysis. In summary, our data demonstrates the usefulness of NIR TD optical imaging in tracking delivered cells, giving insights into the migratory properties of the injected cells. 1. Introduction Optical imaging encompasses several desirable characteristics: it is rapid, noninvasive and nontoxic (it is not based on radiation). For these reasons, it is the optimal tool for performing long-term longitudinal studies in vivo [1], given the possibility to adapt experimental protocols to different fields of investigation. Specifically, time domain (TD) optical imaging technology allows for whole-body near infrared (NIR) fluorescence lifetime analysis, based both on the specificity of fluorescence probes and the sensitivity of their emission lifetime to environmental characteristics [2]. Different kinds of probes can be conjugated with fluorescence dyes: antibodies [3], polysaccharides [4], peptides [5], and also cells can be imaged to evaluate their in vivo biodistribution [6]. At present, clinical optical imaging is an emerging field, and its promising results are supported by preliminary investigations on sentinel lymph node tracers [7] and on peripheral tissue perfusion [8]. In both cases, indocyanine green was used as NIR fluorophore. Moreover, extensive studies have been conducted to confirm sensitivity and tissue diagnostic imaging potentiality of
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