Neural Crest-Derived Dental Pulp Stem Cells arrangement resembles dentinogenesis and contrasts early osteo- genesis. In dentinogenesis, capillaries are in close proximity to odontoblasts and many of these capillaries are fenestrated whereas in osteogenesis the initial bone condensations occur in avascular areas [57–59]. Wnt1-Cre/R26R-LacZ mice demonstrate that DPSCs are neural crest-derived Although we have shown that non-clonal and clonal DPSCs express neural crest genes and differentiate into neural crest- lineages in vitro and form dentin like structures in vivo, the developmental origin of our cultured cells collectively called DPSCs could not be distinguished. Consequently, we used a genetic lineage tracing model, the Wnt1-Cre/R26R-LacZ mouse, to demonstrate the contribution of neural crest to stem cells in dental pulp [9]. In the Wnt1-Cre/R26R-LacZ double-transgenic mice all neural crest derived tissues express reporter driven b-galactosidase. DPSCs isolated from the Wnt1-Cre/R26R-LacZ neonatal mice showed a majority of Wnt1-marked cells in culture stained positive for b-galactosidase with X-gal. Nonetheless, early in culture a minority of cells (approximately 10%) stained negative for X-gal (Figure 9A, arrowheads). Although we could not confirm the origin of this minority, the percentage of b-galactosidase negative cells is consistent with the highest Cre/lox efficiencies reported for other transgenic models [60,61]. To confirm the neural crest origin of DPSCs, we also isolated bone marrow stromal cells (BMSCs) from the same Wnt1-Cre/R26R-LacZ mice and cultured in the same condition. Unlike DPSCs, primary culture of BMSCs at day 8 negatively stained for X-gal, indicating their non-neural crest origin (Figure 9B). A previous study using the same transgenic mouse model, Wnt1-Cre/R26R-LacZ, reported that Figure 9. DPSC clones from Wnt1-Cre/R26R-LacZ mice. (A) DPSCs first isolated from 4–10 day-old Wnt1-Cre/R26R-LacZ showed a majority of Wnt1-marked cells in primary culture on day 8 (d8) which stained positively for b-galactosidase with X-gal. A significant minority (10%) of negative cells was also observed in early cultures (arrowheads). (B) Bone marrow stromal cells (BMSCs) isolated from the same Wnt1-Cre/R26R-LacZ cultured in the same condition for 8 days showed negative staining for X-gal. DPSCs were successfully cloned on day 7–10. Following several passages until day 40 (d40), all clones generated in this experiment (n = 10) were 100% positive for X-gal. (C and D) Two different Wnt1 marked clones were shown. (E) A representative DPSC clone from C57BL6 was negative for X-gal. (F) RT-PCR demonstrated that DPSC clones strongly expressed most of neural crest- related genes, Msi1, Sox10, TrkC, Twist, Snail, and slightly expressed LNGFR, but not in BMSCs. Both DPSCs and BMSCs expressed CD105, a known mesenchymal stem cell marker. DPSC clones also expressed pluripotent stem cell genes, Nanog, Klf4, Sox2 and c-Myc. (G–J) Immunofluorescence showed positive staining of mesenchymal stem cell markers, CD44, CD73, CD105, and neural crest-related marker, MSI1 in DPSC clones (in red). (K) Staining with IgG isotype was used as negative control. DAPI used for nuclei staining is depicted in blue. Scale bars indicate 50 mm. doi:10.1371/journal.pone.0027526.g009 PLoS ONE | www.plosone.org 10 November 2011 | Volume 6 | Issue 11 | e27526