Development of mesenchymal stem cells partially originate from the neural crest Satoru Morikawa a,b , Yo Mabuchi a , Kunimichi Niibe a,b , Sadafumi Suzuki a , Narihito Nagoshi a,c , Takehiko Sunabori a , Shigeto Shimmura d , Yasuo Nagai a , Taneaki Nakagawa b , Hideyuki Okano a , Yumi Matsuzaki a, * a Department of Physiology, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan b Department of Dentistry and Oral Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan c Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan d Department of Ophthalmology, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan article info Article history: Received 6 January 2009 Available online 20 January 2009 Keywords: Mesenchymal stem cells Neural crest Flow cytometry Prospective isolation Developmental origin abstract Mesenchymal stem cells (MSCs) are a heterogeneous subset of stromal stem cells isolated from many adult tissues. Previous studies reported that MSCs can differentiate to both mesodermal and neural lin- eages by a phenomenon referred to as ‘‘dedifferentiation’’ or ‘‘transdifferentiation’’. However, since MSCs have only been defined in vitro, much of their development in vivo is still unknown. Here, we prospec- tively identified MSCs in the bone marrow from adult transgenic mice encoding neural crest-specific P0-Cre/Floxed-EGFP and Wnt1-Cre/Floxed-EGFP. EGFP-positive MSCs formed spheres that expressed neural crest stem cell genes and differentiated into neurons, glial cells, and myofibroblasts. Interestingly, we observed MSCs both in the GFP + and GFP À fraction and found that there were no significant differ- ences in the in vitro characteristics between these two populations. Our results suggest that MSCs in adult bone marrow have at least two developmental origins, one of which is the neural crest. Ó 2009 Elsevier Inc. All rights reserved. Mesenchymal stem cells (MSCs) possess two important proper- ties. First, MSCs have long-term self-renewality, seen as the ability to form fibroblast colony-forming units (CFU-Fs) and to sustain proliferation in vitro. Second, they have multi-lineage differentia- tion potential, which is the ability to give rise to multiple mesen- chymal cell lineages including adipocytes, chondrocytes, and osteocytes [1]. The use of MSCs is already a reality in regenerative medicine, an example of which is the recent success in correcting osteogenesis imperfecta [2]. Because MSCs have been isolated from a low-density mononuclear cell population in humans and other species based on their selective adherence to plastic surfaces in vitro, the knowledge of their in vivo characteristics, such as their development, are unknown. Understanding the developmental ori- gin of stem cells is important for regenerative medicine. In order to make regenerative medicine a reality, we need to mimic the steps of physiological development. Several types of stem cells have been identified in adult tissues. For example, several groups have described multipotent stem cells in the bone marrow (BM), but the developmental origin and differentiation potential of these cells are unknown [3]. These stem cells are reported to generate neuronal cells and glial cells, which are known to originate from neural crest cells. The neural crest is a transient embryonic tissue that originates at the neural folds during vertebrate development. Neural crest cells delaminate from the dorsal neural tube and migrate to various locations, where they differentiate into a vast range of cells, including neurons and glial cells of the peripheral nervous system, smooth muscle cells of the heart and great vessels, and bone and cartilage cells of the face [4]. Recently, many reports demonstrated the presence of neural crest stem cells in various adult tissues and suggest their existence in not only embryo but also adulthood [5,6]. The aim of this study is to identify the origin of MSCs prospec- tively. To this end, we examined BM of double-transgenic mice encoding Protein-0 (P0) and Wnt1 promoter-Cre/Floxed-EGFP, in which neural crest-derived cells express EGFP [7,8]. Prior to this approach, we found that two putative MSC markers, PDGFRa and Sca-1, can be used to isolate a subpopulation of BM mononuclear cells (BMMNCs) with approximately 200,000-fold higher CFU-Fs frequency than unfractionated BM (unpublished data, Morikawa et al., in preparation). We compared MSCs derived from neural crest with those not of neural crest origin by prospectively isolating MSCs from adult P0 and Wnt1 promoter-Cre/Floxed-EGFP mice. Our data present evidence that neural crest-derived cells contribute to a MSC subset, which probably occurs from the embryonic period through 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.01.031 * Corresponding author. Fax: +81 3 5363 3566. E-mail address: penguin@sc.itc.keio.ac.jp (Y. Matsuzaki). Biochemical and Biophysical Research Communications 379 (2009) 1114–1119 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc