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Biochem Biophys Res Commun. 2007 Jun 15;357(4):917-23. Epub 2007 Apr 17.

Isolation of multipotent stem cells from adult rat periodontal ligament by neurosphere-forming culture system.

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  • 1Department of Cellular Physiological Chemistry, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan.


Adult multipotent stem cells have been isolated from various non-neural tissues. Here, we report the isolation of multipotent stem cells from rat periodontal ligament (PDL) using neurosphere-forming culture system. Enzymatically dissociated PDL cells were cultured in serum-free basal medium containing EGF, bFGF, and LIF. Free-floating spheres expressing nestin, GFAP, and vimentin were formed by 7 days of the culture. In addition, spheres expressed mRNA of neural crest-associated transcription factors Twist, Slug, Sox2, and Sox9. PDL-derived spheres differentiated into multinucleated myotubes, NFM-positive neuron-like cells, GFAP-positive astrocyte-like cells and CNPase-positive oligodendrocyte-like cells. Methylcellulose colony-forming assay revealed that a single PDL cell could form a sphere at a frequency of approximately 0.01% of total cells. These data indicate that PDL-derived spheres contained multipotent adult stem cells capable of differentiating into both neural and mesodermal progeny. This is the first report of the isolation of PDL-derived stem cells with primitive neural crest stem cell features.

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