Neural Crest-Derived Dental Pulp Stem Cells Figure 4. Non-clonal and clonal DPSC cultures differentiate into mesenchymal lineages. Treatment of DPSC clones with osteogenic media resulted in different outcomes compared to the differentiation of non-clonal DPSCs. (A–D) Clones did not produce positive cells for BSP and OPN while non-clonal populations showed clusters of positive cells for these markers. (E–J) Non-clonal and all clonal DPSC cultures in osteogenic media stained positively for DMP1, DSP, and OCN. (K) Corresponding to staining results, RT-PCR shows the expression of differentiation genes following treatment of clones with osteogenic media, lack of Bsp, but strong expression of Dmp1 was observed in the differentiation of DPSC clones (Osteogenic D21). (L and M) Non-clonal and 4 out of 5 clones (C5–C8) stained positively for COL II after treatment with chondrogenic media. (N) RT- PCR shows strong expression of chondrogenic markers, Sox9 and Col2a1, in DPSCs cultured in chondrogenic media (Chondrogenic D21). Staining and RT-PCR are all derived from a representative clone (C6). RNA isolated from mouse mandible and femur was used as positive control for gene expression. Scale bars indicate 100 mm. doi:10.1371/journal.pone.0027526.g004 and OPN in osteogenic media (Figures. 4B, 4D), corresponding to low expression of Bsp in differentiated clones (Figure 4K). BSP is the osteoblast-specific protein found in bone matrix and highly secreted by osteoblasts [48]. In contrast, consistent with Dmp1 expression (Figure 4K), non-clonal and all clones in osteogenic media stained positive for DMP1 (Figures 4E, 4F), dentin sialoprotein (DSP) (Figures 4G, 4H) and osteocalcin (OCN) (Figures 4I, 4J); all of which are non-collagenous proteins secreted by odontoblasts and found in dentin matrix [31,49,50]. DSP is particularly considered a dentin-specific protein which is highly expressed by odontoblasts [48].These results suggest the presence of odontoblast-like cells more abundant in clonal differentiations. This osteogenic media has been used to differentiate odontoblast- like cells from dental pulp cells in previous studies, thus it is not surprising that DPSCs can differentiate into odontoblast-like cells when induced with such media [51]. However, the clonal populations showed a more odontogenic than osteogenic pheno- type under this condition. Furthermore, all non-clonal DPSCs and 4 out of 5 DPSC clones stained positively for COL II (Figures 4L, 4M) after treatment with chondrogenic media, which was confirmed by the Sox9 and Col2a1 expression (Figure 4N). In contrast, all non-clonal and clonal DPSC cultures acquired a smooth muscle-like morphology and stained positive for a-smooth muscle actin after treatment with PDGF-BB media (Figures 3F, 5B). In addition, Q-RT-PCR of smooth muscle genes showed higher expression of smooth muscle- and pericyte-related genes (Sm22-alpha, Sma, SMHC, and calponin) in the undifferentiated clonal populations as compared to non- clonal (Figure S6). In turn, the differentiated progeny derived from the clonal populations showed a pattern of smooth muscle maturation with significantly increased levels of myocardin, Sm22-alpha, Sma, SMHC, and calponin (.10 folds higher than non-clonal differentiated cells and .100 folds higher compared to smooth muscle cells) whereas in the non- clonal populations this trend of maturation is not apparent (Figure S6) [52,53]. Three out of five clones stained positive for neurofilament (NF-160/200) (Figure 5D), S100 (Figure 5F), and NG2 (Figure 5H) which are neuronal cytoskeletal, glial, and oligodendrocyte markers, respectively, after induction with neuro- genic media [54]. The NF staining was confirmed with RT-PCR of neurofilament-light (NFL) and -heavy (NFH) (Figure 5I). In conclusion, 3 out 5 clones could differentiate into odontoblast-like, chondrocyte-like, smooth muscle-like and neuronal-like cells, PLoS ONE | www.plosone.org 5 November 2011 | Volume 6 | Issue 11 | e27526