IF analysis for ES transcription factors.

<p>(A–F): FITC labeled antibody was used to evaluate, by IF, Oct4 protein expression in DPSCs cultured in 1.25% HS (A, B), 10% FBS (C, D) and 1.25% C-HS (E, F). (G–L): FITC labeled antibody was used to evaluate, by IF, Sox-2 expression in DPSCs in proliferation in 1.25% HS (G, H) 10% FBS (I, J) and 1.25% C-HS (K, L). (M–R): FITC labeled antibody was used to evaluate, by IF, Nanog expression for DPSCs in proliferation in 1.25% HS (M, N) 10% FBS (O, P) and 1.25% C-HS (Q, R). Bar scales 75 µm.</p

DPSC morphological characterization and growth curve.

<p>(A–F) Isolated DPSC were small, highly proliferative with reduced cytoplasm (A) in 2.5% HS medium, (B) in 1.25% HS medium, (C) in 0.5% HS medium, (D) in 0.25% HS medium. (E) 5×10⁴ DPSCs plated in 60 mm well, in presence of media added with 2.5% HS, 1.25% HS, 0.5% HS, 0.25% HS, and 1.25% HS medium alone, were maintained for 5 days without medium changing and used to generate growth curves. Estimated doubling time, expressed as mean ± SD, for 2.5–1.25% HS was 25.3±1.5 hours and 29.8±1.3 hours respectively. Doubling time for cells in media with 0.5%, 0.25% and 1.25% HS medium alone was 31.4±1.2, 31.4±2, and 146±1 hours respectively. X-DPSCs count Y-medium type, (p<0.05). (F) Morphology of DPSCs isolated and cultured in 1.25% HS medium alone. Bar scales 150 µm. (G–I) After two weeks DPSC isolated in 1.25% HS medium (G) displayed a homogeneous morphology with reduced cytoplasm and were small, highly proliferative with respect to DPSCs cultured in 10% FBS medium (H) and in 1.25% C-HS (I). Bar scales 150 µm. (J) 5×10⁴ DPSCs plated in 60 mm well were maintained for 5 days in culture, with medium changing at day three, and used to generate growth curves. Estimated doubling time, expressed as mean ± SD, was 28±2 hours in 1.25% HS, instead was 45±2.5 hours in presence of 10% FBS and 31.5±2 hours in 1.25% C-HS. X-DPSCs count Y-medium type, (p<0.05).</p

Testing diverse HS percentages.

<p>Testing diverse HS percentages.</p

Telomerase and telomeres.

<p>(A) Graph evidence the relative TRT activity of DPSCs cultured in 1.25% HS medium, 10% FBS and 1.25% C-HS media compared to Ntera2 and positive control (Ctr +). X-absorbance Y-cell types, (p<0.05). (B–G) Dot plot of FL1-height versus FL3-height of cells hybridized with hybridization solution without Telomere PNA Probe. Gates are set around cells in the G0/1- phase for both sample cells, DPSC in 1.25% HS medium (R7) (B), DPSC in 10% FBS (R7) (D) DPSC in 1.25% C-HS medium (R7) (F) and control cells (1301 cell line). Dot plot of FL1-height versus FL3-height of cells hybridized with Telomere PNA Probe/FITC in Hybridization Solution. Gates are set around cells in the G0/1-phase for both sample cells DPSC in 1.25% HS medium (R7) (C), DPSC in 10% FBS (R7) (E) DPSC in 1.25% C-HS medium (R7) (G) and control cells (1301 cell line). The above relative telomere length (RTL) value indicates that the average telomere fluorescence per chromosome/genome in DPSCs in 1.25% HS medium, 10% FBS and 1.25% C-HS was about 18±1.1%, 17±0.9% and 17.1±1.1% respectively of the telomere fluorescence per chromosome/genome in the control cells (1301 cell line) (p<0.05).</p

Telomerase reverse transcriptase activity.

<p>Telomerase reverse transcriptase activity.</p

Cluster differentiation markers expression.

<p>Cluster differentiation markers expression.</p

HS and FBS comparison.

<p>HS and FBS comparison.</p

Osteoblastic differentiation.

<p>Osteoblastic differentiation.</p

Tissue specific primers.

<p>Tissue specific primers.</p

SSEA4, 1 FACS analysis.

<p>SSEA4, 1 FACS analysis.</p