BMSCs did not derive from neural crest [62]. DPSCs were successfully cloned on day 7–10 before the first passage. All DPSC clones (n = 10) stained 100% positive with X-gal (Figures 9C, 9D) and were grown for four passages. Since the majority of cells early in culture stained positive with X-gal, it is unlikely that the homogeneous expression of b-galactosidase in clones derived prior to the first passage resulted from epigenetic changes induced by long-term culture. X-gal staining of C57BL6-derived DPSCs in a late passage was negative (Figure 9E) demonstrating specificity of X-gal staining and ruling out non-specific or mammalian endogenous b-galactosidase activity in the X-gal staining. Like our previous DPSCs, RT-PCR showed that Wnt1-Cre/R26R-LacZ derived DPSC clones expressed pluripotent stem cell genes, Nanog, Klf4, Sox2, and c-Myc, but not Oct4. As expected, DPSCs expressed neural crest-related genes, Msi1, Sox10, TrkC, LNGFR, Twist, and Snail, whereas BMSCs did not express these genes. Twist, Snail, and Slug are expressed by cells in eptithelial-mesenchymal transition (EMT) process during embryological development such as migratory neural crest cells [63]. Previous studies have shown that those EMT-related genes can be expressed by mesenchymal stem cells from bone marrow [64,65]. Both DPSCs and BMSCs also expressed CD105, also known as endoglin, which is a well- known mesenchymal stem cell (MSC) marker (Figure 9F) [66]. In addition, immunofluorescence showed that DPSC clones in our culture condition stained positively for CD44, CD73, CD105, three MSC markers previously reported in human DPSCs, and stained positively for MSI1, a neural crest-related marker (Figures 9G–9K) [67–69] . We corroborated the presence of neural crest-derived MSCs in dental pulp of neonatal mice, Wnt1-Cre/R26R-LacZ and Tie2-GFP that co-expressed MSC and neural crest-related markers. Since neural crest-derived cells of the Wnt1-Cre/R26R-LacZ express b- galactosidase under Wnt1 promoter driven, we studied the expression of MSC markers in Wnt1 marked cells in dental pulp. From all three of MSC markers expressed by cultured DPSCs (CD44, CD73, and CD105), only CD44 is strongly expressed in dental pulp tissue. Staining of tooth sections with anti-b- Neural Crest-Derived Dental Pulp Stem Cells galactosidase showed that many dental pulp cells and all odonto- blast cells in the Wnt1-Cre/R26R-LacZ mouse were positive for b- galactosidase, confirming their neural crest origin (Figure 10A). Cells co-expressing b-galactosidase and CD44 were found in the Wnt1-Cre/R26R-LacZ dental pulp particularly in the sub-odonto- blastic and perivascular regions, indicating DPSCs are of neural crest origin (Figure 10A, arrowheads and arrows, respectively). Tie2-GFP derived dental pulp showed CD44-positive cells in the same areas found in the Wnt1-Cre/R26R-LacZ (Figure 10B, arrowheads and arrows). The CD44 staining of both transgenic mice’s teeth indicates DPSCs are preferentially localized in perivascular niches as suggested in previous studies [70–72]. Additionally, we used another double staining of MSI1 and CD44 to confirm co-expression of CD44, a mesenchymal stem cell marker, and MS1, a neural crest-related marker, by DPSCs. Like the co-staining of b-galactosidase and CD44, DPSCs stained positively for MSI1 and CD44 preferentially located in the same areas; underneath odontoblast cell layer and surrounding blood vessels (Figures 10C, 10D, arrowheads and arrows, respectively). As expected, we did not observe any positive cells for b- galactosidase in the Tie2-GFP derived dental pulp and IgG isotype control staining (Figures 10E, 10F). Accordingly, we observed blood vessels in the Tie2-GFP dental pulp expressing GFP (Figures 10B, 10F, arrowheads). The co-expression of b-galacto- sidase or neural crest-related and MSC markers indicates the existence of neural crest-derived DPSCs in murine neonatal teeth. Next, we determined in vitro multi-differentiation capacity in neural crest lineages of the Wnt1-Cre/R26R-LacZ derived clonal DPSCs using the same differentiation protocols. Cells treated with adipogenic media generated lipid-containing cells positive for Oil Red O (Figure 11A). We performed chondrocyte differentiation in both monolayer and cell pellet methods cultured in chondrogenic media [73,74]. After 21 days differentiated cells in the monolayer secreted extracellular matrix stained positively for COLII (Figure 11B). In addition, the chondrogenic pellet culture conditions induced DPSCs to form aggregations in differentiated medium, but not in stem cell media. These aggregates stained Figure 10. Localization of neural crest-derived stem cells in dental pulp of neonatal mice. (A) Immunofluorescence showed Wnt1-Cre/ R26R-LacZ derived dental pulp cells and odontoblasts expressing b-galactosidase (B-gal, in green). The co-expression of b-galactosidase and mesenchymal stem cell marker, CD44, in plasma membrane (in red) was observed in DPSCs located in sub-odontoblastic (arrowheads) and perivascular regions (arrows). (B) In Tie2-GFP dental pulp only vessels express green fluorescent protein (GFP), CD44 positive cells were particularly located in sub-odontoblastic (arrowheads) and perivascular areas (arrows). (C and D) The co-localization of CD44 (in red) and MSI1 (in pseudo-color cyan), a neural crest-related marker, was observed in cells located in sub-odontoblastic (arrowheads) and perivascular areas (arrows) of both Wnt1- Cre/R26R-LacZ and Tie2-GFP, respectively. (E) Staining with IgG isotypes are shown as negative control. (F) Tie2-GFP derived dental pulp cells did not express b-galactosidase, but blood vessels in this transgenic mouse were labeled by GFP (arrows). D = Dentin, DP = Dental pulp, OD = Odontoblasts. Scale bars indicate 100 mm. doi:10.1371/journal.pone.0027526.g010 PLoS ONE | www.plosone.org 11 November 2011 | Volume 6 | Issue 11 | e27526