Neural Crest-Derived Dental Pulp Stem Cells Figure 5. DPSC clones differentiate into neural crest non-mesenchymal lineages. (B) All clones showed smooth muscle-like cells positive for smooth muscle actin (in red). (D, F, and H) Three clones (C6–C8) showed positive cells for neurofilament (NF-160/200), S100, and NG2. (I) The NF staining was confirmed by RT-PCR of neurofilament-light (NFL) and -heavy (NFH). (A, C, E, and G) Undifferentiated clones were negative for smooth muscle and neuronal markers. DAPI used for nuclei staining is depicted in blue. Scale bars indicate 100 mm. doi:10.1371/journal.pone.0027526.g005 demonstrating their neural crest stem cell capacity (comparison summarized in Table S1). Nevertheless, each clone showed some variability in lineage differentiation capacity and efficiency, which reflects heterogeneity and/or a complex hierarchy of progenitor/ stem cells in our cultures. DPSCs produce dentin-like structure in vivo To determine in vivo differentiation of DPSCs, we transplanted DPSCs labeled with a red fluorescence dye (PKH-26) intramus- cularly and subcutaneously in immune compromised Rag1 mice to avoid immune rejection [55]. We used murine bone marrow stromal cells (BMSCs) cultured and transplanted under the same conditions in order to compare DPSCs in vivo differentiation capacity with another mesenchymal line derived from a different origin (mesodermal). Two-weeks after intramuscular (IM) transplantation, non-clonal DPSCs identified as PKH-26+ (Figure S7A), created compacted collagen bundles as indicated by Masson’s trichrome (Figure S7B). The presence of collagen fibers was confirmed with polarized light microscopy (Figure S7C). Staining for dentin or bone proteins was only slightly positive (data not shown), suggesting that the compacted collagen bundles did not resemble dentin- or bone- like structure. We then hypothesized that longer in vivo transplantation will result in more mature phenotype of the transplanted cells. Nonetheless, 12-week IM transplantations of non-clonal, clonal DPSCs, and BMSCs, only generated abundant immature collagen fibers that stained blue in Masson’s trichrome (Figures S7D-S7F), and polarized light (Figures S7G-S7I) but were negative for dentin or bone proteins (data not shown). This indicates that the skeletal muscle is not an inductive environment for formation of mature mineralized matrix. To explore if DPSCs can form more mature matrix structures in vivo, we used a more permissive model of matrix formation, subcutaneous (SC) transplantation with hydroxyapatite/tricalcium phosphate scaffolds (HAp/TCP) [2]. Here we transplanted three clones separately, which expressed high levels of Nanog and Klf4 by Q-RT-PCR (Figure S5), and showed multi-lineage neural crest differentiation capacity (Table S1). In these transplants we also used BMSCs as control cell line. Five weeks after SC transplantation with HAp/TCP, PKH26- labeled DPSC clones (Figure 6A) generated collagen forming- tissue demonstrated by Masson trichrome (Figure 6C) and polarized light (Figure 6D, white arrowheads). In addition, PKH-26+ clonal cells produced extracellular matrices which showed strong staining for DMP1 (Figures 7A, 7D), and slightly positive staining for DSP (Figures 7B, 7E) and BSP (Figures 7C, 7F). However, strong positive staining of DSP (Figure 7E, black arrowheads) and BSP (Figure 7F, black arrowheads) was observed in the cytoplasm of transplanted cells. DPSC clones were negative for dentin and bone proteins prior to transplantation. All antibodies for dentin and bone matrices showed appropriate staining pattern in tooth sections used as positive control to PLoS ONE | www.plosone.org 6 November 2011 | Volume 6 | Issue 11 | e27526