Neural Crest-Derived Dental Pulp Stem Cells Figure 11. Multi-differentiation capacity in neural crest-derived mesenchymal lineages of Wnt1-marked DPSCs. (A) Double staining showed that X-gal+ treated cells in adipogenic media demonstrated lipid droplets-containing cells positive for Oil Red O. (B) Chondrogenic differentiation by monolayer method showed that a cluster of chondrocyte-like cells derived from DPSCs secreted extracellular matrices positive for COLII. (C and D) DPSCs cultured in chondrogenic media by pellet method formed aggregates containing highly proteoglycan which stained positively for toluidine blue and alcian blue, respectively. (E) Differentiated DPSCs in osteo-odontogenic media stained with IgG isotype as negative control showed X-gal positive staining. (F and G) DPSCs treated in osteo-odontogenic media for 21 days secreted dentin matrices stained positively for DMP1 and DSP; those extracellular matrices were not found in cells cultured in stem cell media. (H–J) RT-PCR from each differentiation confirmed the staining results. DPSCs treated in each differentiation media up-regulated specific adipogenic (A D14), chondrogenic (C D21), and osteo-odontogenic (B D21) genes after 14 to 21-day culture. RNA of mouse adipose tissue, femur, and mandible was used as positive control while the reaction without cDNA was used as negative control. Scale bars indicate 100 mm. doi:10.1371/journal.pone.0027526.g011 positively for toluidine blue and alcian blue, which stain cartilage extracellular matrices (Figures 11C, 11D) [35,73]. For osteo- odontogenic differentiation, in contrast to undifferentiated cells, treated cells with differentiation media showed clusters of cells stained positively for DMP1 and DSP (Figures 11E–11G). The staining results were confirmed by RT-PCR (Figures 11H–11J). The gene expression after each differentiation revealed that induced cells expressed higher levels of specific adipogenic, chondrogenic, and osteo-odontogenic markers, respectively. In addition to mesenchymal lineages, clonal DPSCs gave rise into neuronal-like cells positive for neurofilament and S100, confirming by NFL and NFH gene expression (Figures 12A–12E). DPSCs cultured in smooth muscle media showed smooth muscle- like cells positive for smooth muscle actin; some of which stained positive for smooth muscle myosin heavy chain, calponin, and caldesmon, which are mature markers of smooth muscle cells (Figures 12F–12I) [53]. Q-RT-PCR showed treating DPSCs in smooth muscle media revealed up-regulation of specific gene expression for smooth muscle differentiation, serum response factor (SRF), Sm22-alpha, Sma, SMHC, and calponin (Figure 12J). Thus, Wnt1-marked DPSCs can successfully differentiate into neural crest lineages. Altogether these results confirm that clonal DPSCs generated in our culture conditions are all derived from neural crest origin. Lastly, to determine the differentiation potential of cultured Wnt1-marked DPSCs in vivo, Wnt1-marked DPSCs were subcuta- neously transplanted following the same protocol described above. Transplanted Wnt1-marked DPSCs were identified by anti-b- galactosidase staining in transplants (Figure 13A) whereas HAp/ TCP only and transplanted tissue stained with IgG isotype control were negative for anti b-galactosidase staining (Figures 13B, 13C). Transplanted Wnt1-marked DPSCs were also positive for CD44 and MSI1, two markers expressed by DPSCs in dental pulp tissue and in vitro (Figures 13D, 13E). Wnt1-marked DPSC concentrated near HAp/TCP areas, and secreted dense extracellular matrices. Some transplanted Wnt1- marked DPSCs were elongated, polarized, and arranged per- pendicularly to HAp/TCP, resembling odontoblast-like cells (Figures 13F–13H, arrows). In addition, transplanted Wnt1-marked DPSCs also formed clusters of loose connective tissue containing PLoS ONE | www.plosone.org 12 November 2011 | Volume 6 | Issue 11 | e27526