Isolation and Characterization of Neural Crest-Derived Stem Cells from Dental Pulp of Neonatal Mice Kajohnkiart Janebodin1,4, Orapin V. Horst2, Nicholas Ieronimakis3, Gayathri Balasundaram3, Kanit Reesukumal5, Busadee Pratumvinit5, Morayma Reyes1,3* 1 Department of Oral Health Sciences, School of Dentistry, University of Washington, Seattle, Washington, United States of America, 2 Departments of Dental Public Health Sciences and Endodontics, School of Dentistry, University of Washington, Seattle, Washington, United States of America, 3 Department of Pathology, Institute for Stem Cell and Regenerative Medicine, School of Medicine, University of Washington, Seattle, Washington, United States of America, 4 Department of Anatomy, Faculty of Dentistry, Mahidol University, Bangkok, Thailand, 5 Department of Clinical Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand Abstract Dental pulp stem cells (DPSCs) are shown to reside within the tooth and play an important role in dentin regeneration. DPSCs were first isolated and characterized from human teeth and most studies have focused on using this adult stem cell for clinical applications. However, mouse DPSCs have not been well characterized and their origin(s) have not yet been elucidated. Herein we examined if murine DPSCs are neural crest derived and determined their in vitro and in vivo capacity. DPSCs from neonatal murine tooth pulp expressed embryonic stem cell and neural crest related genes, but lacked expression of mesodermal genes. Cells isolated from the Wnt1-Cre/R26R-LacZ model, a reporter of neural crest-derived tissues, indicated that DPSCs were Wnt1-marked and therefore of neural crest origin. Clonal DPSCs showed multi- differentiation in neural crest lineage for odontoblasts, chondrocytes, adipocytes, neurons, and smooth muscles. Following in vivo subcutaneous transplantation with hydroxyapatite/tricalcium phosphate, based on tissue/cell morphology and specific antibody staining, the clones differentiated into odontoblast-like cells and produced dentin-like structure. Conversely, bone marrow stromal cells (BMSCs) gave rise to osteoblast-like cells and generated bone-like structure. Interestingly, the capillary distribution in the DPSC transplants showed close proximity to odontoblasts whereas in the BMSC transplants bone condensations were distant to capillaries resembling dentinogenesis in the former vs. osteogenesis in the latter. Thus we demonstrate the existence of neural crest-derived DPSCs with differentiation capacity into cranial mesenchymal tissues and other neural crest-derived tissues. In turn, DPSCs hold promise as a source for regenerating cranial mesenchyme and other neural crest derived tissues. Citation: Janebodin K, Horst OV, Ieronimakis N, Balasundaram G, Reesukumal K, et al. (2011) Isolation and Characterization of Neural Crest-Derived Stem Cells from Dental Pulp of Neonatal Mice. PLoS ONE 6(11): e27526. doi:10.1371/journal.pone.0027526 Editor: Songtao Shi, University of Southern California, United States of America Received November 10, 2010; Accepted October 19, 2011; Published November 8, 2011 Copyright: 2011 Janebodin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by funding from National Institutes of Health grant 5R03AR054955 to MR, and the Department of Pathology and Laboratory Medicine, University of Washington. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: morayma@uw.edu Introduction Dental pulp is a loose connective tissue which contains heterogenous cell populations located in the central part of the tooth [1]. Dental pulp stem cells (DPSCs), first discovered by Gronthos and Shi, were isolated from human adult teeth and characterized as highly proliferative cells with self renewal, multi- differentiation in vitro, and capacity to form dentin/pulp-like structures in vivo [2,3]. Initial studies described heterogeneity of DPSCs. Subsequent studies attempted to use clonogenic assays and stem cell markers to purify DPSC populations [3,4]. Nevertheless, the multi-differentiation of DPSCs in vitro and in vivo has been variable as described in several reports [3,5,6]. Differential Notch expression was observed in various locations within the dental pulp. Notch signaling is important for stem cell determination, which implies that dental pulp may harbor several stem cell subpopulations with different capacity and origins [7]. Alternatively all DPSCs share a common developmental origin but their niche and location dictates their behavior. The expression and function of the Eph/ephrin molecules on DPSCs, which play an essential role in the neural crest migration, suggests that neural crest contributes to DPSCs [8]. The Wnt1-Cre/R26R-LacZ used to trace neural crest developmental origin has indicated that the majority of dental pulp cells are Wnt1-marked, with some contribution from unmarked Wnt1- non-neural crest cells [9]. Recently, stem cell populations in adult human dental pulp have been described based on low-affinity nerve growth factor receptor (LNGFR) and b1-integrin expressions, suggesting that the former population is neural crest-derived [10]. In a separate study stem cells isolated from rat embryonic mandibular processes, of neural crest origin, showed multi-differentiation to neural and mesen- chymal lineages suggesting DPSCs retain the differentiation capacity of embryonic progenitors [11]. Collectively, these reports urged us to investigate the origin of DPSCs. Cranial neural crest (CNC) cells migrate to the first branchial arch and participate in differentiation of dental mesenchyme to develop dental pulp and giving rise to dentin-forming cells, odontoblasts [12]. In addition, CNCs show multipotent capacity by giving rise to skeletal and connective tissues of head and neck, as well as, neuron and glial cells in cranial ganglia [13]. During tooth development dental pulp tissue is derived from neural crest and non-neural crest tissues [9]. Although neural crest contributes PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e27526