Neural Crest-Derived Dental Pulp Stem Cells 100 mm. RQ values were normalized by the expression of mouse smooth muscle cells. GAPDH was used for the internal control. Error bars represent 6SEM. doi:10.1371/journal.pone.0027526.g012 blood vessels, resembling dental pulp-like tissue (Figures 13F–13H, arrowheads). As expected, transplanted HAp/TCP without DPSCs tissues were devoid of dense extracellular matrices (Figure 13I). Masson’s trichrome confirmed dense collagen matrix formation in DPSC transplants, but not in HAp/TCP transplants only (Figures 13J–13M). In addition, DPSCs secreted extracellular matrices which were positive for dentin matrix proteins, DMP-1 and DSP (Figures 13N, 13O). The staining of dentin matrices were completely negative in the transplantation of HAp/TCP only (Figures 13P, 13Q). Corresponding to our previous results of DPSC Figure 13. Wnt1-marked DPSCs gave rise to odontoblast-like cells and generated dentin-like structure in vivo. The Wnt1-marked DPSCs were subcutaneously transplanted in HAp/TCP scaffolds and analyzed 5 weeks post-transplantation. (A) anti b-galactosidase staining (in green) indicated the location of transplanted cells in DPSC transplantation. (B and C) Negative staining of anti b-galactosidase were observed in both transplantation of HAp/TCP only and IgG isotype control, respectively. (D and E) Co-localization of cells positive for b-galactosidase (in green) and CD44 (in red), as well as, MSI1 (in green) and CD44 (in red) were shown in odontoblast-like cells surrounding HAp/TCP scaffold surfaces in DPSC transplants. (F–H) H&E staining demonstrated the morphology of transplanted tissues in DPSC transplants. The transplanted DPSCs concentrated near HAp/TCP surfaces, and secreted extracellular matrices. Some of transplanted cells were elongated, polarized, and located perpendicularly to HAp/TCP surfaces, resembling odontoblast-like cells (indicated by arrows). Many loose connective tissue areas containing small blood vessels (indicated by arrowheads) surrounded by transplanted cells were found in DPSC transplants, resembling dental pulp-like tissues. (I) transplantations of HAp/TCP without cells were devoid of dense extracellular matrix. (J–M) Masson’s trichrome staining confirmed the formation of collagen matrices found in DPSC transplants, but not in the transplantation of HAp/TCP only, respectively. (J–L) DPSC transplants showed elongated and polarized transplanted cells (indicated by arrows). (N–Q) DMP and DSP staining was strongly positive in DPSC transplants, but negative in the transplantation of HAp/TCP only, respectively. (O) White arrowheads illustrate polarized transplanted cells positive for DSP. (R) Staining with IgG isotype was used as negative control. DAPI used for nuclei staining is depicted in blue. The dot lines represent a boundary between extracellular matrices secreted by DPSCs and dental pulp-like structure. DP = Dental pulp-like structure, HAp/TCP = Hydroxyapatite/tricalcium phosphate. Scale bars indicate 100 mm. doi:10.1371/journal.pone.0027526.g013 PLoS ONE | www.plosone.org 14 November 2011 | Volume 6 | Issue 11 | e27526