The International Journal of Developmental Biology

Int. J. Dev. Biol. 54: 1287 - 1294 (2010)

Vol 54, Issue 8-9

Induction of neural crest cells from mouse embryonic stem cells in a serum-free monolayer culture

Open Access | Original Article | Published: 11 August 2010

Yuko Aihara1, Yohei Hayashi1, Mitsuhi Hirata2, Nobutaka Ariki3, Shinsuke Shibata4, Narihito Nagoshi4,5, Mio Nakanishi1, Kiyoshi Ohnuma1, Masaki Warashina6, Tatsuo Michiue1, Hideho Uchiyama7, Hideyuki Okano4, Makoto Asashima1,8 and Miho Kusuda Furue*,2

1Department of Life Sciences (Biology), Graduate School of Arts and Sciences, University of Tokyo, Tokyo, 2Laboratory of Cell Cultures, Department of Disease Bioresources, National Institute of Biomedical Innovation, Osaka, 3Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, 4Department of Physiology, Keio University, School of Medicine, Tokyo, 5Department of Orthopedic Surgery, Keio University, School of Medicine, Tokyo, 6Cell Biology Research Center, Genome Research Laboratories, Wako Pure Chemical Industries, Ltd., Hyogo, 7International Graduate School of Arts and Sciences, Yokohama City University, Yokohama, and 8Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, Japan


The neural crest (NC) is a group of cells located in the neural folds at the boundarybetween the neural and epidermal ectoderm. NC cells differentiate into a vast range of cells,including neural cells, smooth muscle cells, bone and cartilage cells of the maxillofacial region, and odontoblasts. The molecular mechanisms underlying NC induction during early developmentremain poorly understood. We previously established a defined serum-free culture condition formouse embryonic stem (mES) cells without feeders. Here, using this defined condition, we havedeveloped a protocol to promote mES cell differentiation into NC cells in an adherent monolayerculture. We found that adding bone morphogenetic protein (BMP)-4 together with fibroblastgrowth factor (FGF)-2 shifts mES cell differentiation into the NC lineage. Furthermore, we haveestablished a cell line designated as P0-6 that is derived from the blastocysts of P0-Cre/Floxed-EGFP mice expressing EGFP in an NC-lineage-specific manner. P0-6 cells cultured using this protocolexpressed EGFP. This protocol could be used to help clarify the mechanisms by which cellsdifferentiate into the NC lineage and to assist the development of applications for clinical therapy.


neural crest, embryonic stem cells, defined serum-free condition, BMP-4

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