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The purpose of this study was to characterize neural crest–derived cells within the adult murine iris. The iris was isolated from P0-Cre/Floxed-EGFP transgenic (TG) mice. The isolated iris cells formed EGFP-positive spheres on non-adhesive culture plates. Immunostaining showed that these EGFP-positive spheres expressed neural crest markers including Sox10 and p75NTR, and these cells showing in vitro sphere-forming ability were originally resided in the iris stroma (IS), in vivo. Real-time RT-PCR showed that the EGFP-positive spheres expressed significantly higher levels of the neural crest markers than EGFP-negative spheres and bone marrow–derived mesenchymal stem cells. Furthermore, the iris stromal sphere had capability to differentiate into various cell lineages including smooth muscle and cartilage. These data indicate that neural crest–derived multipotent cells can be isolated from the murine IS and expanded in sphere culture.