to major cell constituents in dental pulp, the origin of DPSCs is still unclear. Herein, we describe neural crest-derived DPSCs from Tie2- GFP and Wnt1-Cre/R26R-LacZ neonatal mice and demonstrate their highly proliferative capacity and multi-differentiation in neural crest-lineage in vitro and in vivo. Furthermore, the comparison of the niches and mineralized matrix generated by bone marrow stromal cells (BMSCs) and DPSCs when transplanted in vivo illustrates main differences in dentinogenesis and osteogenesis and the role of pericytes and microvessels during these processes. Results DPSC isolation and culture Dental pulp was isolated from neonatal murine mandibular molar teeth because these developing teeth have not formed roots yet (Figures S1A, S1B), which makes pulp dissection feasible. Since the dental pulp is a highly vascularized tissue, the Tie2-GFP mouse model was used to determine the contribution of endothelial and hematopoietic cells in culture by screening for GFP expression driven by the Tie2 promoter [14]. Following constant monitoring for the presence of GFP positive cells we concluded all dental pulp cultures derived from Tie2-GFP were completely negative for GFP, indicating our cultures did not contain endothelial and/or hematopoietic cells (data not shown). In addition, cells harvested from early cultures were negative for endothelial expressed genes vWF (von Willebrand Factor), CD31 (PECAM), angiopoeitin receptors Tie1 and Tie2 and Ve-Cadherin by RT-PCR (data not shown). To identify highly proliferative populations, we cultured freshly isolated dental pulp mononuclear cells at low density, 1000 cells/ cm2, in stem cell media with 2% serum as a selective condition to enrich for stem cell outgrowth, and 5% CO2/O2 to more accurately replicate physiological conditions [15]. Within two days in culture, cells began to proliferate and form colonies (Figure S1C). By day 10 in culture, cells formed large and confluent colonies (Figure S1D), that were split and subsequently passaged approximately every 3–4 days. In each passage, we used a 1:4 dilution as the standard ratio for cell expansion. Cells were cultured until passage 14 (day 90), and grew at a consistent and steady proliferation rate without signs of senescence, suggesting that DPSCs are highly proliferative. DPSCs from three indepen- dent isolations were characterized; each of which demonstrated similar growth pattern and proliferation rate. The morphology of cultured cells was heterogeneous in early culture but most appeared spindle-shaped (Figure S1E). DPSCs express stem cell and neural crest–related genes To gain insights into the stem cell properties and possible origin of DPSCs, we surveyed the expression of stem cell genes as well as neural crest and mesodermal genes in freshly isolated dental pulp tissue and three DPSC lines generated from early to late cultures. RT-PCR (Figure 1A) showed absence of Oct4 in both fresh tissue and cells in early culture, while Sox2 was generally expressed. However, Oct4 was expressed in the late culture (passage 7) by two DPSC lines. Surprisingly, Klf4 was highly expressed and maintained even in late passages by all three DPSC lines. Klf4 and Sox2 are two of four pluripotency genes required to generate inducible pluripotent stem cells [16]. Moreover, Nanog which is important for the maintenance of pluripotency in embryonic stem cells was up- regulated in culture [17]. In turn, expression of these pluripotency genes suggests the presence of a primitive stem cell population in our cultures. DPSCs also expressed variable levels of c-Kit which has been reported to play important roles for cell survival, proliferation and differentiation of multiple types of stem cells including neural crest stem cells destined to form melanocytes [18]. Neural Crest-Derived Dental Pulp Stem Cells Figure 1. Gene profile of DPSC cultures. Gene expression of fresh dental pulp and DPSCs cultured several passages from three isolations (#1, #2 and #3) are shown. (A) Stem cell genes, Oct4, Sox2, Klf4, Nanog and c-Kit, were observed; however, Oct4 was inconsistent and transient from early to late passages. (B) In contrast to neural crest develop- mental genes Gsc and GATA6, early mesodermal developmental genes Brachyury and Mesp2 were not expressed. (C) Neural crest-related genes TrkC, Pdgfra, LNGFR, Twist, Slug, Snail, Msi1, and NCAM, were continuously expressed in all three non-clonal DPSCs. RNA isolated from mouse embryonic stem cells (mESCs) and salivary gland were used as positive control for the expression of stem cell/early developmental genes and neural crest genes, respectively. The PCR reaction without template was used as negative control. doi:10.1371/journal.pone.0027526.g001 To characterize the potential origin of DPSCs, we examined by RT-PCR the expression of early developmental genes in DPSCs (Figure 1B). As expected, we observed a complete absence of genes expressed by mesodermal cells during development, Brachyury and Mesp2 [19,20]. DPSCs did however express genes associated with neural crest development, Goosecoids (Gsc) and GATA6 [21,22]. In addition, DPSCs expressed embryonic neural crest and neuronal stem/precursor cell associated genes (Figures 1C, S2), tyrosine kinase C (TrkC), platelet-derived growth factor receptor-alpha (Pdgfra), LNGFR, Twist, Snail, Slug, Sox10, neural cell adhesion molecule (NCAM), and Musashi1 (Msi1) [23–27]. We further examined the presence of neural crest associated proteins in DPSCs by immunocytochem- istry. Undifferentiated cells showed positive staining for the pluripotent transcription factor, KLF4 and neural crest-related factors, MSI1 and SOX10 (Figure S2). Although we cannot rule out the existence of non-neural crest cells in these non-clonal cultures, these results suggest that a neural crest derived population existed in our DPSC cultures. DPSCs in vitro differentiate into neural crest-lineage cells During development of head and neck, neural crest cells migrate to the craniofacial region and give rise to neurons, glia, PLoS ONE | www.plosone.org 2 November 2011 | Volume 6 | Issue 11 | e27526