Figure 2.

SHED possessed stem-cell characteristics. (A–E) The remnant pulp showed STRO-1 (open arrows in A) and CD146 (open arrows in B) immunopositive staining for cells in perivascular areas. Fluorescence-activated cell-sorting analysis showed that ex vivo-expanded SHED contained ≈9% STRO-1-positive cells (C). SHED expressed STRO-1 (D) and CD146 (E) (arrows). (F–I) SHED expressed the osteogenic and angiogenic markers ALP, MEPE, bFGF, and endostatin. (J and K) SHED were either cultured with regular medium (J) or l-ascorbate-2-phosphate, dexamethasone, and inorganic phosphate for 4 weeks (K). Alizarin red staining showed mineralized nodule formation in the induction (K). (L) Western blot analysis showed an up-regulated expression of CBFA1, ALP, MEPE, bone sialoprotein (BSP), and DSPP after the induction as described above. HSP90 was used to assess the amount of protein loaded per sample. (M) Human recombinant BMP-4 (300 ng/ml, 24 h) was added to induce a significant up-regulation of CBFA1, Osterix, and Osteocalcin (OC) in SHED as detected by semiquantitative PCR.