BrdU assay showed that, at 48?h, E8-cultured hDPSCs exhibited a stronger proliferation capacity with higher fluorescence labeling rate than culture with SCM (Fig

BrdU assay showed that, at 48?h, E8-cultured hDPSCs exhibited a stronger proliferation capacity with higher fluorescence labeling rate than culture with SCM (Fig.?3dCf) ( em p /em ? ?0.01). indicated that this expression of PPAR-, RUNX2, OCN and?MAP-2?was higher in E8 group.? Conclusions Compared with serum-containing medium, E8 medium exhitibed higher ability in maintaining the cell proliferation, pluripotency, migration, and stability. This new serum-free culture environment might be applicable for hDSC culture in the future. test. Statistical significance was accepted at em p /em ? ?0.05. Results Changes in cell morphology Cells cultured Chromafenozide in SCM proliferated sparsely in a single layer and exhibited common spindle Chromafenozide and polygonal shapes. On the other hand, cells cultured in E8 tended to grow in close contact with one another and demonstrated more homogeneous shapes (Fig.?1). Cells cultured in E8 for 48?h and 96?h did not present differences in cell morphology. Open in a separate windows Fig. 1 Cell morphology. a Images of primary culture for 14 d and 28?d. b-d Differences in cell morphology after culture in E8?(left) and serum-containing medium (right;?SCM; DMEM +?5% FBS) for b 24?h, c 48?h, and d 96?h Identification of MSC surface markers Both the SCM group and the E8 group expressed high levels of CD29, CD44, CD73, CD90, and CD166, and did not express CD31, CD45, or CD105 (Fig.?2), which agreed with MSC surface marker expression and proved that the majority of these cells were DPSCs. Open in a separate windows Fig. 2 Characterization of?hDPSCs surface markers?by flow cytometry. The red curves are the blanks. The blue curves are the E8 or SCM. E8 can promote hDPSC proliferation CFU-F results indicated that, at 10 days, a significant difference was observed between E8 and SCM (Fig.?3aCc) ( em p /em ? ?0.01). BrdU assay showed that, at 48?h, E8-cultured hDPSCs exhibited a stronger proliferation capacity with higher fluorescence labeling rate than culture with SCM (Fig.?3dCf) ( em p /em ? ?0.01). We used CCK-8 to analyze hDPSCs cultured for 4?h, 24?h, 48?h, 72?h, 96?h, 120?h, and 144?h. Data were obtained as average optical density (OD) values and a CCK-8 growth curve was produced (Fig.?3i) Statistical differences were observed between the E8 group and the SCM group at 24?h, 48?h, 72?h, and 96?h ( em p /em ? ?0.01). Open in a ARHGEF11 separate windows Chromafenozide Fig. 3 Colony-forming unit fibroblasts (CFU-F) of a serum-containing medium (SCM) and b E8. Statistical analysis of c CFU-F comparison ( em n /em ?=?5) and d bromodeoxyuridine (BrdU) proliferation assay ( em n /em ?=?5). BrdU fluorescence of hDPSCs in?e E8 and f SCM. g Cell cycles were analyzed with FlowJo software. h Statistical analysis of the cell cycle ( em n /em ?=?5). i Cell proliferation analysis using the CCK-8 assay. The different optical density (OD) values are presented at 4?h, 24?h, 48?h, 72?h, 96?h, 120?h, and 6 days ( em n /em ?=?10). * em p /em ? ?0.05, ** em p /em ? ?0.01 To study why cell?proliferation rate differed?between E8 and SCM, we analyzed the cell cycle and apoptosis. Images captured by FlowJo software are presented in Fig.?3g. A significant difference was seen, and E8-cultured hDPSCs possessed fewer cell numbers in the G0/G1 ratio ( em p /em ? ?0.01) and higher numbers in the S ratio ( em p /em ? ?0.01) and G2/M ratio ( em p /em ? ?0.01) (Fig.?3h). Flow cytometry was used to analyze apoptosis, and the resultshowed difference between the SCM group and the E8 group regarding early ( em p /em ? ?0.05), late ( em p /em ? ?0.01), and total Chromafenozide apoptosis (p? ?0.01) (Fig.?4c). Images processed by FlowJo software are also presented in Fig.?4a. Western blotting and immunofluorescence also exhibited that this apoptosis rate of hDPSCs in E8 group was lower than that in SCM group (Figs.?4b and ?and5c).5c). Altogether, it can be deduced that this E8 medium increased the hDPSC proliferation rate through accelerating the cell splitting velocity and decreasing the cell apoptosis rate. Open in a separate windows Fig. 4 Cell apoptosis assay and Western blot. a Representative images of cell apoptosis from both E8 and serum-containing medium (SCM) groups. b Western blot images of cell apoptosis from both E8 and SCM groups. c Cell apoptosis comparison of the two groups ( em n /em ?=?5). d Western blot of DMP1 and DSPP (for odontogenic markers), OPN, RUNX2, and ALP (osteogenic markers), and GAPDH set as control. * em p /em ? ?0.05, ** em p /em ? ?0.01 Open in a separate Chromafenozide window Fig. 5 Immunofluorescence?of hDPSCs. a OCT4, SOX2, and NANOG for cell pluripotency. b DMP1 and DSP1-H for odontogenic tendency. c p53 images of cell apoptosis from both the E8 and serum-containing medium (SCM) groups. d OCN, OPN, RUNX2, and BMP2 for the osteogenic tendency Expression of stem cell markers by immunofluorescence and Western blotting Immunofluorescence was applied to analyze OCT4, SOX2, and NANOG for.