Techawattanawisal Wanida, Nakahama Kenichi, Komaki Motohiro, Abe Mayumi, Takagi Yuzo, Morita Ikuo
Department of Cellular Physiological Chemistry, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan.
Biochem Biophys Res Commun. 2007 Jun 15;357(4):917-23. doi: 10.1016/j.bbrc.2007.04.031. Epub 2007 Apr 17.
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.
成人多能干细胞已从各种非神经组织中分离出来。在此，我们报告使用神经球形成培养系统从大鼠牙周膜（PDL）中分离出多能干细胞。将酶解的PDL细胞在含有表皮生长因子（EGF）、碱性成纤维细胞生长因子（bFGF）和白血病抑制因子（LIF）的无血清基础培养基中培养。培养7天后形成了表达巢蛋白、胶质纤维酸性蛋白（GFAP）和波形蛋白的游离球体。此外，球体表达神经嵴相关转录因子Twist、Slug、Sox2和Sox9的mRNA。PDL来源的球体分化为多核肌管、神经丝蛋白（NFM）阳性的神经元样细胞、GFAP阳性的星形胶质细胞样细胞和2',3'-环核苷酸3'-磷酸二酯酶（CNPase）阳性的少突胶质细胞样细胞。甲基纤维素集落形成试验表明，单个PDL细胞形成球体的频率约为总细胞数的0.01%。这些数据表明，PDL来源的球体含有能够分化为神经和中胚层后代的多能成体干细胞。这是首次报道分离出具有原始神经嵴干细胞特征的PDL来源干细胞。
