Neural Crest-Derived Dental Pulp Stem Cells Figure 6. Subcutaneous transplantation of DPSCs and BMSCs. Non-clonal and clonal DPSCs (C6–C8) were labeled with PKH-26 before transplantation to track cells in vivo. Panel A shows 5-week subcutaneous (SC) transplantation of DPSC clones with HAp/TCP. (A and B) PKH-26+ cells SC transplanted with HAp/TCP were identified in bright red fluorescent color whereas the HAp/TCP scaffold without cells was devoid of red fluorescent cells despite some autofluoresence derived from HAp/TCP. The HAp/TCP scaffold without cells did not show mineralized tissues (data not shown). Most of transplanted cells surrounded HAp/TCP. (C) These clonal cells generated collagen-forming matrix shown in blue of Masson’s trichrome. (D, white arrowheads) Polarized light was used to confirm collagen formation. Panel B shows 12-week SC transplantation of non-clonal DPSCs, clonal DPSCs, and BMSCs with HAp/TCP. (E–G) H&E staining showed the morphology of tissues created by non-clonal, clonal DPSCs and BMSCs. Capillaries and small blood vessels near mineralized matrices were found in DPSC transplants. In contrast, BMSCs formed bone condensations near HAp/TCP devoid of vessels. In BMSC transplants capillaries were found mainly in the adipose tissue. The black arrowheads indicate the location of capillaries and small blood vessels. (H–J) Masson’s trichrome showed the intensive staining of collagenous extracellular matrices generated by both DPSCs and BMSCs. (K–M) polarized light images with high magnification of transplanted tissues showed different collagen arrangement produced by DPSCs and BMSCs. (K and L, white arrowheads, and M) The white arrowheads indicate the direction of polarized collagen fibers created by DPSCs run perpendicularly to HAp/TCP while that created by BMSCs do not run perpendicularly to scaffold surface. DAPI used for nuclei staining is depicted in blue. HAp/TCP = Hydroxyapatite/tricalcium phosphate. Scale bars indicate 200 mm. doi:10.1371/journal.pone.0027526.g006 confirm the antibody specificity (Figure S8). We observed that these mineralized tissues formed by DPSCs contained abundant microvessels that stained positive for CD31, an endothelial marker (Figure S9A). We consistently observed some PKH-26+ (donor DPSCs) cells adjacent to these microvessels. These PKH-26+ cells seem scattered around vessels. Staining for a-smooth muscle actin revealed that these microvessels did not contain a thick muscle layer but were wrapped by pericyte-like cells that were SMA positive and some were also PKH-26+ (Figures S9C, S9D, S9F). Many of these microvessels seem fenestrated and stained positive for VEGF receptor 3 (Figures S9E, S9F) [56]. Interestingly, the 12-week SC transplantation showed different composition of mineralized matrices depending on the donor cells, DPSCs and BMSCs. The H&E and Masson’s trichrome showed PLoS ONE | www.plosone.org 7 November 2011 | Volume 6 | Issue 11 | e27526