Mesenchymal stem cell properties of SHED. (A-E) SHED showed a similar osteogenic differentiation potential to BMMSCs. After one week culture induction under osteogenic conditions, ALP activity and numbers of ALP positive cells in SHED and BMMSCs were significantly higher than that of the control SHED and BMMSCs, respectively, by ALP staining (Representative of n = 5) (A) and flow cytometric analysis (Representative of n = 3) (B). Meanwhile, immunoblot analysis showed that the osteogenic induction elevates expression levels of ALP, Runx2, DSP, and OCN in SHED and BMMSCs (C) (***P < 0.001, n = 5). β-actin was used as an internal control. After four weeks culture induction in osteogenic medium, SHED showed increased capacity of forming mineralized nodules as assessed by alizarin red staining (Representative of n = 5). (D). Alizarin red-positive area corresponding to total area was averaged from five independent groups (E). (F-H) SHED showed reduced potential of differentiating into adipocytes compared to BMMSCs. Three weeks post adipogenic induction, lipid accumulation in SHED was less than that in BMMSCs by Oil-red O staining (Representative of n = 5). (F). Number of oil-red O-positive (Oil-Red-O+) cells was calculated as a percentage to total cells and averaged from five independent cultures (G) (*P < 0.05). Immunoblot assay indicated that SHED expressed lower levels of adipocyte-specific molecules LPL and PPARγ than BMMSCs at three weeks post adipogenic culture (H). Three independent assays showed the similar results. (I-K) SHED were capable of forming mineralized tissue when transplanted subcutaneously into immunocompromised mice using HA/TCP as a carrier (Representative of n = 3). (I). It appeared that SHED form similar amounts of mineralized tissue as seen in a BMMSC transplant (Representative of n = 3) (I, J), but they generated significantly less bone marrow elements than BMMSCs (K). Newly formed mineralized tissue and bone marrow areas were calculated as a percentage of the total area and averaged from three independent transplant assays (***P < 0.001). B = bone, BM = bone marrow, C =: connective tissue, H =: hydroxyapatite and tricalcium carrier. (L-P) SHED and BMMSCs express multiple signaling pathways during culture expansion at passage 3. SHED and BMMSCs expressed TGFβ receptor I and II, Smad 2 and phosphorylated Smad 2 (L); P38, phosphorylated P38, ERK, and phosphorylated ERK (M); Akt and phosphorylated Akt (N); N-cadherin and β-catenin (O); PDGF receptor and Ang-1 (P). Representative image of n = 5.