Isolation and Culture of Human Stem Cells from Apical Papilla under Low Oxygen Concentration Highlight Original Properties

Abstract

: Stem cells isolated from the apical papilla of wisdom teeth (SCAPs) are an attractive model for tissue repair due to their availability, high proliferation rate and potential to differentiate in vitro towards mesodermal and neurogenic lineages. Adult stem cells, such as SCAPs, develop in stem cell niches in which the oxygen concentration [O₂] is low (3-8% compared with 21% of ambient air). In this work, we evaluate the impact of low [O₂] on the physiology of SCAPs isolated and processed in parallel at 21% or 3% O₂ without any hyperoxic shock in ambient air during the experiment performed at 3% O₂. We demonstrate that SCAPs display a higher proliferation capacity at 3% O₂ than in ambient air with elevated expression levels of two cell surface antigens: the alpha-6 integrin subunit (CD49f) and the embryonic stem cell marker (SSEA4). We show that the mesodermal differentiation potential of SCAPs is conserved at early passage in both [O₂], but is partly lost at late passage and low [O₂], conditions in which SCAPs proliferate efficiently without any sign of apoptosis. Unexpectedly, we show that autophagic flux is active in SCAPs irrespective of [O₂] and that this process remains high in cells even after prolonged exposure to 3% O₂.

Keywords: BNIP3; CD49f; SCAPs; SSEA4; autophagy; differentiation; low oxygen concentration; proliferation.

Figure 1

Design of the experiments. Diagram of the different experimental procedures (EXP I, II, III) is shown with the percentage of O₂ for tooth harvesting, stem cells from apical papilla (SCAP) isolation and expansion. In Experiment (EXP) III, two teeth from the same individual were harvested and processed at 21% or 3% O₂. SCAPs derived from EXP III were named: UBx-SCAP. SCAPs at 21% O₂ were named: UBx-SCAP-N1, N2 and N3. SCAPs at 3% O₂ were named: UBx-SCAP-H1, H2 and H3.

Figure 2

Proliferative advantage of UBx-SCAP isolated under 3% O₂ in comparison with ambient air (21% O₂). (A) At each passage of SCAPs from EXP III, 0.4 (under 3% O₂) or 0.8 (under 21% O₂) millions of cells were seeded in a 75 cm² flask and counted after three or four days. Cumulative population doublings (CPD) were plotted for each individual refered to UBx-SCAP-N1, N2 and N3 (21% O₂) and UBx-SCAP-H1, H2 and H3 (3% O₂), up to 65 days. (B) The mean of time of population doubling for the first 10 passages, for each individual at 21% and 3% O₂ is plotted with standard deviation. Statistical analyses were done with a Mann-Whitney test. ** p < 0.01. *** p < 0.001.

Figure 3

Clonogenicity tests. Plots of the number of colony forming units obtained after two weeks of culture at 21% O₂, for 50 UBx-SCAPs initially isolated and grown at either 21% or 3% O₂ with a low (<7) or high (>10) number of passages, as indicated. Statistical analyses were done with a Mann Whitney test. ** p < 0.01. *** p < 0.001. **** p < 0.0001.

Figure 4

Protein expression analysis. SCAPs from EXP III (early passages of UBx-SCAP) were grown at 3% or 21% O₂. (A) Graphs showing flow cytometry analysis of different markers as indicated ((% of positive cells, left column) and mean of fluorescence intensity (MFI, in arbitrary units, right column)). Numbers 1, 2, 3 refer to the three individuals. For each group, three to six samples from different early passages were analyzed. Statistical analyses were done with a Mann Whitney test. * p < 0.05. (B) Western blot analysis of UBx-SCAP-1 (early passage) grown under 21% or 3% O₂ and of human iPSCs (induced pluripotent stem cells, IMR90 cell line) used as a positive control for expression of Oct4 and Nanog. ERK2 was used as the loading control.

Figure 5

Differentiation of SCAPs. SCAPs from EXP III initially isolated and grown at 3% or 21% O₂ (from early (A) or late passages (B)) were cultured at the same oxygen concentration in control or differentiation media. Representative pictures of experiment performed with UBx-SCAP-3 are shown: Alcian blue (chondrogenic differentiation), red oil (adipogenic differentiation, shown by arrows) and OsteoImage (osteogenic differentiation revealed by green fluorescent dots of HA—hydroxyapatite deposits) staining are shown. Cells from early and late passages are shown as indicated. Scale bar is 100 µm.

Figure 6

Characterization of the autophagy process. (A) Early and (B) late passages of SCAPs, isolated and expanded either at 21% O₂ or at 3% O₂, or isolated and expanded at 21% and switched for four days at 3% O₂, were labelled with anti-LC3 or anti-BNIP3 antibodies as indicated. For each patient and condition cells were treated (+chl) or not (−chl) for 5 h with 20 µM chloroquine before labeling. Representative pictures of UBx-SCAP-3 are shown here. Careful analyses of DAPI stained nuclei did not reveal fragmented chromatin, a feature of apoptosis. Scale bar is 25 µm.

Figure 7

Summary of the salient features of UBx-SCAP harvested, isolated and expanded under 3% O₂ compared with SCAP at 21% O₂ (e.g., EXP III). We have developed a unique new model of adult teeth-derived stem cells (obtained from three individuals) and characterized their properties under different O₂ concentrations, towards potential applications in regenerative medicine. SCAPs at 21% O₂ were named: UBx-SCAP-N1, N2 and N3 and SCAPs at 3% O₂ were named: UBx-SCAP-H1, H2 and H3.