Neural Crest-Derived Dental Pulp Stem Cells Figure 7. Immunohistochemistry of subcutaneous DPSC and BMSC transplanted tissues. Panel A shows the staining of 5-week subcutaneous (SC) clonal DPSC transplanted tissues. (A–C) Three DPSC clones (C6–C8) secreted extracellular matrices which were positive for DMP1, slightly positive for DSP and BSP by immunoperoxidase staining. (D–F) High magnification images correspond to the rectangular areas of A–C. (D) Strong DMP1 staining was seen in the matrices. (E and F, arrows) Strong positive staining for DSP and BSP were observed in the transplanted cells. Panel B shows the staining of 12-week SC transplantations of non-clonal DPSCs, clonal DPSCs, and BMSCs with HAp/TCP. (G–I) Only HAp/TCP transplantation without donor cells did not show any extracellular matrices positive for dentin and bone proteins. (J–O) Both non-clonal and clonal DPSCs secreted extracellular matrices which were strongly positive, but different intensity, to DMP1, DSP, and BSP. All insets in each figure show high magnification images of the transplanted tissues to illustrate the morphology and positive intracellular staining of transplanted cells. (K, M, and N insets) transplanted cells were elongated, polarized, and showed very close relation to scaffold surface, resembling odontoblast-like cell morphology. (K, L, M, N, and O insets) Positive staining of DMP1, DSP, and BSP was seen in transplanted DPSCs. Some cells showed elongation and polarization. (P, arrowheads and arrows) BMSC transplanted cells showed osteoblast-like cells (arrowheads) lining bone surface, and osteocyte-like cells (arrows) in lacunae which stained positively for DMP1. (Q) Negative staining for DSP was observed in bone matrix and transplanted BMSC cells. (R, inset) Positive staining for BSP was observed in bone matrix secreted by transplanted BMSCs. (S–V) Transplanted sections stained with only anti-rabbit biotinylated antibody or IgG isotype were used as negative control. HAp/TCP = Hydroxyapatite/tricalcium phosphate. Scale bars indicate 200 mm. doi:10.1371/journal.pone.0027526.g007 bone-like structure created by BMSCs (Figures 6G, 6J), but not in non-clonal and clonal DPSC transplantations (Figures 6E, 6F, 6H, and 6I). The structures produced by both DPSC populations demonstrated collagen-forming matrices in many areas that were arranged perpendicularly to the surface, which was confirmed using polarized light (Figures 6K, 6L). Corresponding to previous studies, the DPSC transplantation showed that elongated cells created collagen fibers which run perpendicularly to HAp/TCP surfaces, resembling the morphology and arrangement of odontoblasts (Figures 6K, 6L, white arrowheads) [2,3,50]. In contrast, in the BMSC transplantation we observed osteocyte-like cells trapped in matrix of collagen fibers which did not run perpendicularly to the mineralized matrix (Figures 6J, 6M) [2,3]. As negative control, the SC transplantation with only HAp/ TCP, did not show any positive staining of DMP1, DSP, and BSP, respectively (Figures 7G–7I). Conversely, immunohistochemistry PLoS ONE | www.plosone.org 8 November 2011 | Volume 6 | Issue 11 | e27526