transplantation, these observations confirmed that Wnt1-marked DPSCs generate odontoblast-like cells, dentin-like matrix and dental pulp-like tissue in vivo. In addition to odontoblast-like cell differentiation, we found that some of DPSCs were strongly positive for smooth muscle actin, and those were located adjacent to blood vessels close to HAp/ TC, suggesting pericyte-like phenotype (Figures S10A–S10F, arrowheads). DPSC-derived pericyte-like cells were also found surrounding blood vessels distant to HAp/TCP (Figures S10G– S10I, arrowheads). Nevertheless, some pericytes found in DPSC transplants were not derived from transplanted DPSCs as they did not stain positive for anti b-galactosidase. This transplantation model demonstrated that Wnt1-marked DPSCs were able to give rise not only odontoblast-like cells, but also pericyte-like cells. Discussion Differences in DPSC proliferation and differentiation capacities in vitro and in vivo as described in previous reports indicate heterogeneity within DPSC populations [3,6,75]. In turn, such heterogeneity may be attributed to distinct developmental origins. The hypothesis that DPSCs are of neural crest origin has been suggested and indirectly related by previous reports [8–11]. Accordingly, human dental pulp contains self-renewing DPSCs capable of giving rise to mesenchymal-lineages and non-mesen- chymal cells such as neuron-like, smooth muscle-like, and melanocyte-like cells, indicating that these cells might be derived from neural crest [3,76,77]. Herein we explored several criteria to determine if stem cells in dental pulp originate from neural crest. First we investigated the proliferative capacity of murine DPSCs cultured in stem cell selective media, as well as, the expression of stem cell and neural crest genes. Next, we determined in vitro and in vivo multi-differentiation of DPSCs into neural crest-lineage. Lastly, we used the Wnt1-Cre/R26R-LacZ transgenic mouse to conclusively determine that DPSCs generated in our cultures are of neural crest origin. Unlike human, mouse DPSCs have not been well described possibly due to the difficult nature of isolating dental pulp from murine teeth [2]. A recent study characterized progenitors and stem cells in dental pulps of mouse molars in uneruption and eruption states in vitro and revealed a small population of mesenchymal multipotent cells in erupted molars but not in the dental pulp of the unerupted molars. Instead, a majority of dental pulp cells of the unerupted molar were osteo-dentogenic progenitor cells [78]. In our studies, we determined the existence of stem cells in mouse dental pulp from unerupted molars which exhibit high proliferation and multi-differentiation into mesenchymal and non-mesenchymal lineages. DPSCs sufficiently self-perpetuated more than 14 passages (,3 months) without morphological changes. DPSCs chosen for all differentiation experiments were harvested near passage 7, the time which Nanog and Klf4 expression was highest (Figure S5). In this study we used the optimal conditions for long-term cultivation of stem cells without any signs of degeneration or spontaneous differentiation; near physiological oxygen concentration, and a low concentration of serum supplemented with platelet derived growth factor-BB (PDGF-BB), epidermal growth factor (EGF), insulin transferrin selenium (ITS), dexamethasone, and leukemia inhibitory factor (LIF) [15,79]. This combination differs from previously described culture condition used to grow human and mouse DPSCs [2,78]. Therefore, a direct comparison of culture conditions must be undertaken to determine if DPSC described here are equivalent to previously described populations. Interestingly the expression of embryonic pluripotent genes suggests the existence of primitive stem cells within the DPSC Neural Crest-Derived Dental Pulp Stem Cells population that share a similar genetic program to embryonic and inducible pluripotent stem cells [16]. Q-RT-PCR results showed down-regulation of Klf4 and Nanog after differentiation of DPSCs, suggesting that both transcription factors play a similar role and function for maintenance of DPSC multi-potentiality and undifferentiated state. Consistent with our hypothesis of a neural crest origin, DPSCs expressed multiple neural crest markers whereas they did not show mesodermal developmental markers. To determine that DPSCs fulfill the stem cell criteria, in addition to self-renewal, we also explored their multi-differentiation capacity with emphasis on neural crest lineages. Explants of cranial and trunk neural crest cells give rise to chondrocytes, glia, neuronal cells, smooth muscle cells, and melanin-forming cells, which are all neural crest-lineage derived during embryogenesis [13]. In our differentiation conditions, treated DPSCs expressed markers for osteoblast, chondrocyte, neuron, smooth muscle and adipocyte differentiation, indicating that non-clonal DPSCs contains multipotent stem cells with capacity to differentiate into mesenchymal and non-mesenchymal lineages of neural crest cells. Consistently human DPSCs can in vitro differentiate into non-mesenchymal lineages [3,76]. DPSC clones, initiated from early passages of non-clonal cultures, were sub-cultured for 1 month without changing cell morphology. Like non-clonal DPSCs, all clones expressed Klf4, Nanog, and neural crest-related genes, suggesting that clones maintain neural-crest multipotential. Our observations showed DPSC clones gave rise to odontoblast-like, chondrocyte-like, adipocyte-like, smooth muscle-like, and neuronal-like cells. To confirm the neural crest origin of DPSCs, we used a genetic lineage tracing model of neural crest derivatives. Although the Wnt1-Cre/R26R-LacZ mouse has been widely used to study neural crest development, to date the origin of DPSCs has not been examined [9]. Staining results of Wnt1-Cre/R26R-LacZ tooth sections showed the existence of neural crest-derived DPSCs in sub-odontoblastic and perivascular regions. Those two areas have been reported as stem cell niches in dental pulp tissue [70–72]. Under our culture conditions, early dental pulp cells isolated from the Wnt1-Cre/R26R-LacZ revealed the majority of cells were of neural crest origin. This is consistent with the study by Chai et al (2000) showing that dental pulp consists of cells from neural crest and non-neural crest origins [9]. In addition, Wnt1-Cre/R26R- LacZ-derived BMSCs, previously known as non-neural crest- derived used as a control showed negative X-gal staining [62]. The X-gal staining of both DPSCs and BMSCs indicates that the Wnt1-Cre/R26R-LacZ is a good strategy to study the developmental origin of DPSCs. This model definitely confirms the neural crest origin of DPSCs. In spite of a mixed cell population of DPSC in early culture, after 6-week-in vitro expansion, all cells were positive by X-gal staining indicating only neural crest-derived cells remained and were present in late cultures. The Wnt1-Cre/R26R-LacZ-derived DPSC clones also expressed strongly pluripotent stem cell and neural crest-related genes, and gave rise to neural crest lineages. In addition to the expression of neural crest genes and differentiation capacity into neural crest-lineage, this lineage tracing data undeniably proves that in our culture condition DPSCs are neural crest-derived stem cells. To further assess DPSC plasticity, we transplanted cultured DPSCs to examine their response in vivo in two different models: subcutaneous (SC) and intramuscular (IM) transplantations. Interestingly, each transplantation method resulted in different cell fates indicating that DPSCs are not under a fixed program as environmental factors influence their response. Short subcutane- ous transplantation with HAp/TCP resulted in the production of a PLoS ONE | www.plosone.org 15 November 2011 | Volume 6 | Issue 11 | e27526