and HAp/TCP, respectively. DAPI used for nuclei staining is depicted in blue. BV = Blood vessel, HAp/TCP = Hydroxyap- atite/tricalcium phosphate. Scale bars indicate 100 mm. (TIF) Table S1 Variable differentiation capacity of non-clonal and clonal DPSCs. Table S1 shows the summary and comparison of the capacity of non-clonal DPSCs and clones to differentiate into neural crest-derived mesenchymal (osteogenic, odontogenic, adipogenic, and chondrogenic) and non-mesenchy- mal (smooth muscle and neuronal) lineages. These data indicate that non-clonal DPSCs were able to give rise to all neural crest- lineages. In contrast, DPSC clones differentiated at passage 7 showed differentiation capacity into certain lineages, but lack osteogenic and adipogenic capacity. Nonetheless, the clones still showed multi-differentiation capacity into mesenchymal and non- mesenchymal lineages. Interestingly, DPSC clones isolated from the Wnt1-Cre/R26R-LacZ differentiated at passage 4 show the capacity to differentiate into osteoblasts and adipocytes, which differs from that of our previous clones. This may indicate DPSCs lose differentiation capacity to certain lineages during long-term cultures. In addition, differences in the differentiation capacity among the clones indicate heterogeneity or hierarchical relation- ship of stem cells and progenitors. Note that POS (positive) means that cells can give rise to that lineage whereas NEG (negative) means that cells did not differentiate into that lineage. (DOC) The mouse-specific primer sequences. Table S2 (DOC) References 1. Trowbridge, HO (2003) Pulp biology: progress during the past 25 years. Aust Endod J 29: 5–12. 2. Gronthos, S, Mankani M, Brahim J, Robey PG, Shi S (2000) Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A 97: 13625–13630. 3. Gronthos, S, Brahim J, Li W, Fisher LW, Cherman N, et al. (2002) Stem cell properties of human dental pulp stem cells. Journal of Dental Research 81: 531–535. 4. Shi, S, Gronthos S (2003) Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp. Journal of Bone and Mineral Research 18: 696–704. 5. d’Aquino, R, Graziano A, Sampaolesi M, Laino G, Pirozzi G, et al. (2007) Human postnatal dental pulp cells co-differentiate into osteoblasts and endotheliocytes: a pivotal synergy leading to adult bone tissue formation. Cell Death Differ 14: 1162–1171. 6. Huang, GT, Shagramanova K, Chan SW (2006) Formation of odontoblast-like cells from cultured human dental pulp cells on dentin in vitro. Journal of Endodontics 32: 1066–1073. 7. Lovschall, H, Tummers M, Thesleff I, Fuchtbauer EM, Poulsen K (2005) Activation of the Notch signaling pathway in response to pulp capping of rat molars. European Journal of Oral Sciences 113: 312–317. 8. Stokowski, A, Shi S, Sun T, Bartold PM, Koblar SA, et al. (2007) EphB/ephrin- B interaction mediates adult stem cell attachment, spreading, and migration: implications for dental tissue repair. Stem Cells 25: 156–164. 9. Chai, Y, Jiang X, Ito Y, Bringas P, Jr., Han J, et al. (2000) Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis. Development 127: 1671–1679. 10. Waddington, RJ, Youde SJ, Lee CP, Sloan AJ (2009) Isolation of distinct progenitor stem cell populations from dental pulp. Cells Tissues Organs 189: 268–274. 11. Zhang, J, Duan X, Zhang H, Deng Z, Zhou Z, et al. (2006) Isolation of neural crest-derived stem cells from rat embryonic mandibular processes. Biol Cell 98: 567–575. 12. Thesleff, I (2003) Epithelial-mesenchymal signalling regulating tooth morpho- genesis. Journal of Cell Science 116: 1647–1648. 13. Abzhanov, A, Tzahor E, Lassar AB, Tabin CJ (2003) Dissimilar regulation of cell differentiation in mesencephalic (cranial) and sacral (trunk) neural crest cells in vitro. Development 130: 4567–4579. 14. Motoike, T, Loughna S, Perens E, Roman BL, Liao W, et al. (2000) Universal GFP reporter for the study of vascular development. Genesis 28: 75–81. 15. Ma, T, Grayson WL, Frohlich M, Vunjak-Novakovic G (2009) Hypoxia and stem cell-based engineering of mesenchymal tissues. Biotechnol Prog 25: 32–42. Neural Crest-Derived Dental Pulp Stem Cells Table S3 (DOC) Table S4 (DOC) Lists of differentiation media. Lists of antibody for staining. Text S1 Supporting Information Methods and Support- ing Information Reference. (DOC) Acknowledgments We thank Dr. Michael Cunningham (University of Washington) for the Wnt1-Cre/R26R-LacZ mice and his review of this manuscript, Dr. Kevin Tompkins (Chulalongkorn University) for training the pulp isolation, Drs. Thomas Morton Jr. and Ron Seifert (University of Washington) for their useful comments in histology, Dr. Martha Somerman (University of Washington) and Dr. Thanaphum Osathanon (Chulalongkorn University) for cDNA and tooth sections, Dr. Guenter Daum and Dr. William Mahoney Jr. (University of Washington) for Q-RT-PCR primers and cDNA in smooth muscle differentiation experiments. We further thank Drs. Brian Foster, Kanako Nagatomo, Jeremy Horst, Yuji Shiba, Jill Weyers, and also thank Jirawan Wade, Jianxun Sun, Sarah Park, Ambika Gunaje, and Oanh Mai for research technical expertise and useful comments. Author Contributions Conceived and designed the experiments: MR KJ. Performed the experiments: KJ NI GB. Analyzed the data: MR NI KJ. Contributed reagents/materials/analysis tools: OH BP KR. Wrote the paper: KJ MR. 16. Takahashi, K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, et al. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131: 861–872. 17. Boyer, LA, Lee TI, Cole MF, Johnstone SE, Levine SS, et al. (2005) Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122: 947–956. 18. Yoshida, H, Kunisada T, Grimm T, Nishimura EK, Nishioka E, et al. (2001) Review: melanocyte migration and survival controlled by SCF/c-kit expression. J Investig Dermatol Symp Proc 6: 1–5. 19. Rashbass, P, Cooke LA, Herrmann BG, Beddington RS (1991) A cell autonomous function of Brachyury in T/T embryonic stem cell chimaeras. Nature 353: 348–351. 20. Saga, Y, Hata N, Koseki H, Taketo MM (1997) Mesp2: a novel mouse gene expressed in the presegmented mesoderm and essential for segmentation initiation. Genes Dev 11: 1827–1839. 21. Gaunt, SJ, Blum M, De Robertis EM (1993) Expression of the mouse goosecoid gene during mid-embryogenesis may mark mesenchymal cell lineages in the developing head, limbs and body wall. Development 117: 769–778. 22. Nemer, G, Nemer M (2003) Transcriptional activation of BMP-4 and regulation of mammalian organogenesis by GATA-4 and -6. Dev Biol 254: 131–148. 23. Tessarollo, L, Tsoulfas P, Martin-Zanca D, Gilbert DJ, Jenkins NA, et al. (1993) trkC, a receptor for neurotrophin-3, is widely expressed in the developing nervous system and in non-neuronal tissues. Development 118: 463–475. 24. Takakura, N, Yoshida H, Ogura Y, Kataoka H, Nishikawa S, et al. (1997) PDGFR alpha expression during mouse embryogenesis: immunolocalization analyzed by whole-mount immunohistostaining using the monoclonal anti- mouse PDGFR alpha antibody APA5. J Histochem Cytochem 45: 883–893. 25. Morrison, SJ, White PM, Zock C, Anderson DJ (1999) Prospective identifica- tion, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells. Cell 96: 737–749. 26. Meulemans, D, Bronner-Fraser M (2005) Central role of gene cooption in neural crest evolution. J Exp Zool B Mol Dev Evol 304: 298–303. 27. Tomita, Y, Matsumura K, Wakamatsu Y, Matsuzaki Y, Shibuya I, et al. (2005) Cardiac neural crest cells contribute to the dormant multipotent stem cell in the mammalian heart. J Cell Biol 170: 1135–1146. 28. Gronthos, S, Graves SE, Ohta S, Simmons PJ (1994) The STRO-1+ fraction of adult human bone marrow contains the osteogenic precursors. Blood 84: 4164–4173. 29. Ducy, P, Zhang R, Geoffroy V, Ridall AL, Karsenty G (1997) Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 89: 747–754. 30. Nakashima, K, Zhou X, Kunkel G, Zhang Z, Deng JM, et al. (2002) The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 108: 17–29. PLoS ONE | www.plosone.org 20 November 2011 | Volume 6 | Issue 11 | e27526