Characterization of SHED in comparison to BMMSCs. (A) Flow cytometric analysis of cultured SHED at passage 3 revealed expression of STRO-1 (12.06%), CD146 (48.39%), SSEA4 (85.40%), CD73 (91.93%), CD105 (6.77%), CD166 (63.65%), but was negative for surface molecules CD34 and CD45. SHED express high levels of STRO-1 and CD146 (n = 5; P < 0.05) and low level of CD105 (n = 5; P < 0.01) compared to expression levels of STRO-1 (8.36%), CD146 (31.19%), and CD105 (13.27%) in BMMSCs. These signals are shown as the red area. Solid lines indicate signals for isotype matched control antibodies. M1 window show the positive expression defined as the level of fluorescence greater than 99% of the corresponding isoytpe-matched control antibodies. Representative histograms are shown among five donors. (B) Immunoblot analysis confirmed expression of CD73, CD105 and CD166 in SHED and BMMSCs. Representative images of n = 5 donors are presented as results. (C) Immunofluoresence confirmed that SHED express STRO-1, CD146, and SSEA4 along with negative for CD34 and CD45. Red fluorescence indicates the expression of cell surface markers. Blue cell nuclei were stained by DAPI. Images were representative data of independent experiment (n = 5) with consistent results (Bar = 50 μm). (D) SHED were able to form significantly high numbers of single colonies than BMMSCs when 1 × 106 cells were plated at a low density (*P < 0.05) and cultured for 10 days. (E) The proliferation rates of SHED and BMMSCs were assessed by co-culture with BrdU for 18 hours. The number of BrdU-positive cells was presented as a percentage of the total number of cells counted from five replicate cultures. SHED showed a significantly higher proliferation rate in comparison to BMMSCs (**P < 0.01). (F) SHED showed a high activity of telomerase compared to BMMSCs assessed by real time PCR. HEK293T cells (239T) were used as a positive control and heat inactivated 293T (H.I.) cells were used as a negative control. The activity was indicated by a PCR cycle threshold and averaged from three replicated cultures (***P < 0.001).