Neural Crest-Derived Dental Pulp Stem Cells bone, cartilage, and connective tissues, including odontoblasts [9,13]. Therefore, in vitro differentiation of DPSCs into neural-crest lineages supports the hypothesis that DPSCs maintain embryonic neural crest potential. We studied the capacity of DPSCs to differentiate into neural crest-lineage, specifically cranial neural crest derived tissues. Differentiation in mesenchymal lineages. DPSCs exposed to osteogenic, adipogenic, and chondrogenic media for 21 days stained positive for proteins, differentiation markers (Figures 2A– 2K), and expressed transcripts (Figure 2L) specific to each respective differentiation lineage. Immunoperoxidase staining of DPSCs cultured in osteogenic media showed cytoplasmic pattern of bone sialoprotein (BSP) and osteopontin (OPN) in differentiated cells, but not in undifferen- tiated cells (Figures 2A–2D) [28]. RT-PCR showed that dif- ferentiated cells expressed osteogenic markers (Figure 2L, Osteo- genic differentiation); runt related transcription factor 2 (Runx2), osterix (Osx), Opn, Bsp and dentin matrix protein1 (Dmp1) [29,30]. OPN, BSP and DMP1 proteins are present in mineralized tissues such as bone, dentin, and cementum [31,32]. These results suggest that DPSCs cultured in osteogenic media differentiate into osteoblast/ odontoblast-like cells. Small clusters of lipid-containing cells were first observed on day 7 in cells exposed to adipogenic media (data not shown) [33]. Treated cells showed expression of peroxisome proliferator-activated receptor gamma (Pparg2) and CCAAT/enhancer binding protein alpha (CEBPa), which are transcription factors for adipocyte differenti- ation, as well as, leptin and adipsin, which are specific markers of Figure 2. DPSC cultures differentiate into neural crest-derived mesenchymal lineages. (A–D) After 21 days in osteogenic media, differentiated DPSCs were positive for BSP and OPN in a membranous pattern. (F) Cultures in adipogenic media showed lipid droplets-containing cells positive for Oil Red O. (H and J) Cultures in chondrogenic media showed cells in clusters positive for toluidine blue and COL II. (K) Mineralized nodules were observed in cells treated with the chondrogenic media after 21 days. The inset depicts a mineralized nodule in high magnification. (L) RT-PCR confirmed the staining results. For osteogenic differentiation (Osteogenic D21), RT-PCR showed expression of osteoblast- associated genes Runx2, Osx, Opn, Bsp, and Dmp1. For adipogenic differentiation, induced cells (Adipogenic D21) expressed Pparg2 and CEBPa, adipogenic transcription factors, as well as Leptin and Adipsin, markers of adipocytes. For chondrogenic differentiation, treated cells (Chondrogenic D21) expressed chondrocyte-associated genes Sox9 and Col2a1. (A, C, E, G, and I) Confluent undifferentiated cells cultured in stem cell media at day 21 (UD D21) expressed some of differentiation genes, but were negative by staining for all differentiation markers and Oil Red O. Cells were counterstained with hematoxylin. RNA isolated from mouse calvarial bone, adipose tissue, and femur was used as positive control for gene expression. Scale bars indicate 100 mm. doi:10.1371/journal.pone.0027526.g002 PLoS ONE | www.plosone.org 3 November 2011 | Volume 6 | Issue 11 | e27526