AbstractSeveral investigations point out that the volatile fraction of metabolites, often called volatilome, might signal the difference processes occurring in living beings, both in vitro and in vivo. These studies have been recently applied to stem cells biology, and preliminary results show that the composition of the volatilome of stem cells in vitro changes along the differentiation processes leading from pluripotency to full differentiation. The identification of pluripotent stem cells is of great importance to improve safety in regenerative medicine avoiding the formation of teratomas. In this paper, we applied gas chromatography and gas sensor array to the study of the volatilome released by mice transplanted with human induced pluripotent stem cells (hiPSCs) or embryoid bodies (EBs) derived from hiPSCs at 5 days and spontaneously differentiated cells at 27 day. Gas chromatography analysis finds that, in mice transplanted with hiPSCs, the abundance of 13 volatile compounds increases four weeks after the implant and immediately before the formation of malignant teratomas (grade 3) become observable. The same behaviour is also followed by the signals of the gas sensors. Besides this event, the gas-chromatograms and the sensors signals do not show any appreciable variation related neither among the groups of transplanted mice nor respect to a placebo population. This is the first in vivo observation of the change of volatile metabolites released by human induced pluripotent stem cells and hiPSCs-derived cells during the differentiation process. These results shed further light on the differentiation mechanisms of stem cells and suggest possible applications for diagnostic purposes for an early detection of tumor relapse after surgery.