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

https://doi.org/10.1387/ijdb.103173ya

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 Nagoshi^4,5, Mio Nakanishi¹, Kiyoshi Ohnuma¹, Masaki Warashina⁶, Tatsuo Michiue¹, Hideho Uchiyama⁷, Hideyuki Okano⁴, Makoto Asashima^1,8 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

Abstract

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.

Keywords

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