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. 2012;7(11):e48945.
doi: 10.1371/journal.pone.0048945. Epub 2012 Nov 14.

Isolation and characterization of human dental pulp derived stem cells by using media containing low human serum percentage as clinical grade substitutes for bovine serum

Federico Ferro  1 , Renza Spelat, Antonio Paolo Beltrami, Daniela Cesselli, Francesco Curcio
Affiliations

Isolation and characterization of human dental pulp derived stem cells by using media containing low human serum percentage as clinical grade substitutes for bovine serum

Federico Ferro et al. PLoS One. 2012.

Abstract

Adult stem cells have been proposed as an alternative to embryonic stem cells to study multilineage differentiation in vitro and to use in therapy. Current culture media for isolation and expansion of adult stem cells require the use of large amounts of animal sera, but animal-derived culture reagents give rise to some questions due to the real possibility of infections and severe immune reactions. For these reasons a clinical grade substitute to animal sera is needed. We tested the isolation, proliferation, morphology, stemness related marker expression, and osteoblastic differentiation potential of Dental Pulp Stem Cells (DPSC) in a chemically defined medium containing a low percentage of human serum, 1.25%, in comparison to a medium containing 10% Fetal Bovine Serum (FBS). DPSCs cultured in presence of our isolation/proliferation medium added with low HS percentage were obtained without immune-selection methods and showed high uniformity in the expression of stem cell markers, proliferated at higher rate, and demonstrated comparable osteoblastic potential with respect to DPSCs cultured in 10% FBS. In this study we demonstrated that a chemically defined medium added with low HS percentage, derived from autologous and heterologous sources, could be a valid substitute to FBS-containing media and should be helpful for adult stem cells clinical application.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. DPSC morphological characterization and growth curve.
(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).
Figure 2
Figure 2. IF analysis for ES transcription factors.
(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.
Figure 3
Figure 3. IF for ES membrane markers.
FITC labeled antibody was used to evaluate, by IF, SSEA4 expression in DPSCs cultured in 1.25% HS (A), 10% FBS (B) and 1.25% C-HS (C) in expansion condition. DPSCs were reacted with FITC labelled anti-SSEA3 antibody evidencing its expression in 1.25% HS (D), 10% FBS (E) and 1.25% C-HS (F) media. Alkaline Phosphatase assay demonstrates higher enzymatic activity in DPSC cultured in 1.25% HS (G) with respect to DPSCs cultured in 10% FBS (H) and 1.25% C-HS (I). TRITC labeled antibodies were used to evaluate, by IF, TRA-1–60 expression in DPSCs cultured in 1.25% HS (J), 10% FBS (K), 1.25% C-HS (L) and TRA-1–81 in DPSCs cultured in 1.25% HS (M), 10% FBS (N) and 1.25% C-HS (O). SSEA-1 immuno-reactivity was not evident in both culture conditions 1.25% HS (P), 10% FBS (Q) and 1.25% C-HS (R), using TRITC labeled antibody. Bar scales 75 µm.
Figure 4
Figure 4. Telomerase and telomeres.
(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).
Figure 5
Figure 5. Testing and comparing osteoblastic differentiation capacity.
(A–C) DPSCs cultured in 1.25% HS (A), 10% FBS (B) and 1.25% C-HS (C) were osteo-induced for three weeks, and during this period DPSCs changed their morphology, developing an asymmetric shape with an enlarged end. Bar scales 150 µm. (D–I) Real Time PCR showed a comparable increased expression for all the tested markers with no significant differences deriving from the different proliferation conditions, both after one and three weeks of differentiation: 1.25% HS (blue), 10% FBS (red), 1.25% C-HS (green). X- relative expression folds Y- osteo-induction days.
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The work has been supported by Agenzia Spaziale Italiana (ASI) through the MoMa Project. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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