adipocytes (Figure 2L, Adipogenic differentiation) [33]. Despite Pparg2 and leptin expression in undifferentiated cells, Oil Red O- positive lipid-containing adipocytes were observed only in dif- ferentiated cells but not in undifferentiated cells (Figures 2E–2F). DPSCs cultured in chondrogenic media were first observed to display changes from spindle-shaped cells to clusters of round and cuboidal cells representing chondrocyte-like morphology on day 10 [34,35]. After 21 days, these cells secreted matrix with high proteoglycan content which stained positive for toluidine blue (Figure 2H) and collagen type II (COL II) (Figure 2J) and formed calcified nodules (Figure 2K), indicating that these cells form cartilage matrix [36]. RT-PCR demonstrated that treated cells in chondrogenic media expressed Sox9 and increased expression of collagen type II (Col2a1) genes which are specific markers for chondrocyte differentiation [37]. We did not observe these chondrogenic hallmarks in undifferentiated cells despite chondro- genic gene expression in such cells (Figure 2L, Chondrogenic differentiation). RT-PCR showed chondrogenic and adipogenic markers in cells treated with osteogenic media; however, COL II- and/or Oil Red O-positive cells were not observed in this culture (data not shown). Interestingly, we found BSP- and OPN- positive cells, as well as Oil Red O positive-adipocytes in the chondrogenic media (data not shown). This is consistent with previous studies that showed BMP2 is an inducer of not only chondrogenic differentiation but also osteoblastic and adipogenic differentiations [38]. Osx, Bsp, Dmp1, Pparg2, leptin, Sox9, and Col2a1, were expressed in confluent undifferentiated cells in stem cell media at day 21 (Figure 2L), suggesting these are default differentiation pathways of DPSCs in confluent cultures. Alternatively, it has been previously demon- strated that bone marrow stromal cells display an osteogenic imprinting program and express messengers of these proteins in undifferentiated cells [39]. Intriguingly, Q-RT-PCR showed that Nanog and Klf4 were down-regulated during differentiation of DPSCs (Figure S3), suggesting that like embryonic stem cells, these pluripotent genes play a role in maintaining DPSC stemness and thus are down- regulated during differentiation [40]. Differentiation in non-mesenchymal lineages. After exposing DPSCs to neurogenic media, differentiated cells stained positive for N-CAM, a neuronal adhesion molecule, and c- aminobutyric acid (GABA), an inhibitory neurotransmitter pro- duced by GABAergic neurons (Figures 3A–3D) [41,42]. We also treated DPSCs with PDGF-BB to induce smooth muscle differen- tiation [43]. Following day 21 of PDGF-BB, DPSCs showed smooth muscle-like morphology, and stained positive for a-smooth muscle actin (Figures 3E, 3F). However, we could not observe any positive cells of either neurogenic or smooth muscle differentia- tions in the undifferentiated cells (Figures 3A, 3C, and 3E). DPSCs generate single cell colonies Colony-forming capacity is a characteristic growth pattern of many adult stem cells including bone marrow mesenchymal stem cells, and has been used as an indicator of self-renewal [44]. Indeed, a single stem cell should create a colony of progeny cells with all or some daughter cells identical to the original cell. Initially, we attempted to demonstrate colony formation from single DPSC by Fluorescent Activated Cell Sorting (FACS) mediated single cell deposition. Unfortunately, this approach was unsuccessful possible due to sensitivity of DPSCs to cell sorting. Thus we utilized clonal rings commonly used to generate single cell colonies from mesenchymal stem cells [45]. We first seeded cells at several dilutions and determined that the limiting dilution 2 to obtain single colonies was 50–100 cells/cm . After 24 hrs Neural Crest-Derived Dental Pulp Stem Cells Figure 3. DPSC cultures differentiate into neural crest non- mesenchymal lineages. (B and D) Neuronal-induced cells stained positively for neuronal markers; N-CAM ( in green), and GABA (in red). (F) DPSCs in smooth muscle differentiation media showed smooth muscle-like phenotype that stained positively for smooth muscle actin (in red). (A, C, and E) Undifferentiated cells in stem cell media at day 21 did not stain positively for any of the three differentiation markers. DAPI used for nuclei staining is depicted in blue. Scale bars indicate 100 mm. doi:10.1371/journal.pone.0027526.g003 adherent single cells were marked and monitored everyday for the formation of colonies. At day 10, we observed 12 colonies in 600 cm2 (four 150 cm2 petri dishes) (Figures S4A-S4D). After clonal isolation, we cultured these clones in several passages and 5 of 12 survived and proliferated for further characterization. Like the non-clonal populations, RT-PCR of clones expressed the pluripotency genes, Klf4 and Nanog, and neural crest developmental genes, but not mesodermal developmental genes (Figure S4E). Compared to non-clonal populations, clones defini- tively expressed higher and consistent levels of Pax3, a cardiac neural crest developmental gene [46]. All clones demonstrated strong expression of neural crest and neural precursor genes including Vimentin (Figure S4F). Vimentin is expressed in mesen- chymal cells irrespective of their origin or in cells undergoing epithelial-mesenchymal transition such as migratory neural crest cells [47]. Additionally, the clones showed variable Dmp1 but not Dspp, indicating an undifferentiated state of dental pulp cells. In vitro differentiation of DPSC clones To determine if DPSC clones show multi-differentiation in neural crest lineages, we performed in vitro differentiation by exposing 5 clones to the same differentiating media used to differentiate the non-clonal populations. Prior to differentiation, we analyzed Q-RT- PCR levels for Klf4 and Nanog compared to non-clonal populations and dental pulp tissue (Figure S5). In turn, Nanog and Klf4 expression were variable among the clones but comparable to the non-clonal populations. Thus, we assumed that clonal populations should exhibit same differentiation capacity and fate as compared to non- clonal populations (Figures 4A–4J, 4L and 4M). Unlike differentiation of non-clonal cultures (Figures 4A, 4C), we did not observe any differentiated clonal cells positive for BSP PLoS ONE | www.plosone.org 4 November 2011 | Volume 6 | Issue 11 | e27526