Growth patterns and morphological characteristics
Comparative analysis was performed to assess the ability of three different culture systems (one conventional serum-based and two serum/xeno-free cGMP approaches) to support DPSC and aBMMSC initial culture establishment from pulp and osseous biopsies, respectively, and subsequent cell expansion up to p.10. Figure 1a shows the PDT (in days) of each cell type/culture condition from p.2 onward up to p.10; Fig. 1b illustrates the cumulative PD numbers from initial culture establishment (p.0) up to p.10.
Calculation of the PDT at each passage (starting with the same plating density of 5000 cells/cm2 for all cell types/culturing conditions and counting cell numbers at the beginning/end of each passage) showed no statistically significant increase in PDT up to p.10 in CCM, StemMacs and StemPro-expanded DPSCs (p ≥ 0.9997, p ≥ 0.8158 and p ≥ 0.9815, respectively) and in CCM and StemPro-expanded aBMMSCs (p ≥ 0.9157 and p ≥ 0.2992, respectively) (Fig. 1a). In contrast, aBMMSCs expanded with StemMacs showed no significant increase in PDT up to p.6 (p ≥ 0.2658), but from p.7 onward significant delays in cell growth were observed (p < 0.0001 for p.6 vs p.7; p = 0.0312 for p.7 vs p.8; p < 0.0001 for p.8 vs p.9; p = 0.0323 for 9 vs 10; Fig. 1a). This was evidenced by substantially increased PDTs of StemMacs-expanded aBMMSCS, as compared with those expanded with the other two culture media. In two out of six StemMacs-expanded aBMMSC cultures, cells proliferated extremely slowly after p.7.
The initial time required from biopsy uptake to p.1 (performed at 70–80% cell confluency of p.0) was 10.3 (2.1), 10.9 (3.2) and 9.2 (1.9) days for CCM, StemMacs and StemPro-expanded DPSCs, respectively, with no significant differences across culture medium groups (p ≥ 0.7558), indicating in general similar isolation efficiency potential. The respective values for aBMMSCs were 13.4 (3.2), 14.3 (3.9) and 12.9 (2.5) days, failing to reach statistical significance for any of the media used (p ≥ 0.8258). The time (in days) for reaching p.1 was overall significantly lower for DPSCs compared to aBMMSCs for all culture media (p = 0.0279). It should be noted that some (1/6 for CCM, 2/6 for StemMacs and 1/6 for StemPro) aBMMSC cultures failed to reach p.1 after 18 days, due to the low initial cell yield or the very slow growth rate, and were thus excluded from the study.
Regarding cumulative PD numbers (Fig. 1b) no significant differences were found among the DPSCs expanded under the three different culturing systems at any passage (p ≥ 0.6969). After 10 passages, DPSCs reached 39.8 (2.44), 37.6 (1.90) and 38.9 (1.28) cumulative PDs for CCM, StemMacs and StemPro-expanded cells, respectively; these being completed after 46.3 (2.1), 46.9 (3.2) and 45.2 (1.9) days, correspondingly. In contrast, aBMMSC groups showed no significant differences (p ≥ 0.4971) in cumulative PDs up to p.7, whereas from p.8 onward StemMacs-expanded aBMMSCs showed significantly lower cumulative PDs, compared to those expanded with CCM (p < 0.0001 at p.10) or StemPro (p = 0.0026 at p.10) (Fig. 1b). After 10 passages, aBMMSCs reached 30.2 (1.36), 23.9 (6.5) and 28.9 (2.64) cumulative PDs for CCM, StemMacs and StemPro-expanded aBMMSCs, respectively; these being completed 49.4 (3.2), 66.3 (7.6) and 48.9 (2.5) days following initial seeding. No substantial differences in cumulative PD numbers were found between DPSC and aBMMSC cultures up to p.3, whereas from p.4 onward all DPSC culture media groups had significantly higher cumulative PD numbers compared to all respective aBMMSC groups (p values at each passage are shown in Fig. 1b).
Another important observation was that the methodology presented in this study for initial culture establishment and subsequent cell expansion is able to produce a cell yield of approximately 30 million DPSCs after completion of p.2 and approximately 1 billion DPSCs (if the expansion continues without discarding any part of the population) after completion of p.3; the respective values for aBMMSCs are 10 million and 30 million, respectively.
Evaluation of cell morphological characteristics under phase-contrast microscopy (Fig. 2a, b) revealed that serum-expanded DPSCs and aBMMSCs presented noticeable population heterogeneity, consisting of spindle-shaped to stellate-like cells of different sizes, with protrusions of varying number and length; this diversity in phenotype was evident up to late passages. Overall, DPSC cultures consisted of cells considerably smaller in size compared to aBMMSCs; however, they contained several larger cells, seen both at early and late passages, possibly indicating that an intrinsic heterogeneity exists in the cell population. In contrast, DPSC and aBMMSC cultures expanded with both serum-free systems showed a very homogeneous phenotype comprising well-aligned, slender and spindle-shaped cells. This morphology, however, was not maintained at late passages, where a high proportion of flattened, senescent-like cells with multiple intracellular filaments became evident. This was mostly prominent in StemMacs-expanded aBMMSC cultures (Fig. 2b), in accordance with the growth/kinetics data (Fig. 1a, b)
Flow cytometric analysis of cell size vs cell internal complexity (granularity) distribution profiles (FSC vs SSC fluorescence intensity plots; Fig. 2c, d) showed a progressive increase in cell size and granularity with passaging in all types of cultures, but more pronounced in StemMacs-expanded aBMMSC cultures (Fig. 2d), also in conformity with the growth/kinetics data (Fig. 1a, b).
Immunophenotypic profiles
Figure 3 demonstrates representative findings of a single DPSC and aBMMSC culture for each expansion medium at early, middle and late passages. Additional file 1 contains a full panel of representative flow cytometry diagrams of these markers in DPSC cultures at early, middle and late passages.
Immunophenotypic analysis of DPSC (n = 6 donors/culture medium) and aBMMSC (n = 4 donors/culture medium) cultures at p.2–3, p.6–7 and p.10–11 revealed that both types of cells, expanded under either serum-based or serum-free conditions, exhibited very high expression (>95% of the population) of the MSC markers CD90, CD73, CD81 and CD49f/a6-integrin at early passages that was maintained unaltered (CD90, CD73) or slightly but not statistically significantly downregulated (CD81, CD49f) at late passages. These findings were consistent for all cell donors (n = 6/culture system for DPSCs; n = 4/culture system for aBMMSCs) and all three expansion systems (CCM, StemMacs, StemPro). Other markers such as CD105 (>90% DPSCs; > 80% aBMMSCs) and CD146 (>80% DPSCs; > 70% aBMMSCs) were also highly expressed at early passages, but were substantially downregulated at the middle or late passages depending on cell type and donor. Noteworthy, downregulation of these markers was significantly more prominent at the middle to late passages of serum-free (StemMacs, StemPro) expanded cultures, and of smaller magnitude in the serum-based expansion of both DPSCs and aBMMSCs.
Less abundant expression was observed at early passages (p.2–3) for the MSC marker STRO-1 (15.2 (17.7), 4.2 (2.1) and 5.6 (3.2)% for DPSCs and 3.6 (2.3), 3.2 (1.4) and 7.6 (3.8)% for aBMMSCs expanded with CCM, StemMacs and StemPro, respectively) and the embryonic markers SSEA-1 (21.9 (5.3), 23.6 (3.2) and 18 (4.2)% for DPSCs and 3.7 (1.1), 5.2 (1.3) and 7.3 (2.8)% for aBMMSCs expanded with CCM, StemMacs and StemPro, respectively) and SSEA-4 (69.1 (10.6), 52.2 (13.2) and 55.3 (11.3)% for DPSCs and 45.5 (9.6), 20.2 (8.2) and 32.6 (9.8)% for aBMMSCs expanded with CCM, StemMacs and StemPro, respectively). The aforementioned values represent means of six DPSC and four aBMMSC donors evaluated in three independent experiments for each donor. Of paramount importance was that some of the aforementioned markers (CD146, CD105, STRO-1, SEEA-1, SSEA-4) were substantially downregulated with passaging, but more pronounced in the serum-free compared to the serum-based expanded DPSC and aBMMSC cultures (except SSEA-1 that showed similar trends in all cultures). Despite the interindividual variations observed in total % expression of these markers, these patterns were consistent for both cell types and all three expansion media, as well as in all donor biopsies used for cell culture establishment.
The initial very low (<5%) expression of CD34 found at early passages in most DPSC and aBMMSC cultures increased gradually during prolonged culture expansion. Although this finding was inconsistent with all cell types/donors/media, it was noted in several cultures and seemed to be donor related; the latter requiring further investigation. Finally, the hematopoietic stem cell markers CD45 and CD117/c-kit and the embryonic marker SSEA-3 were not expressed (<1%) in any cell types/donors/media during the entire expansion period.
Statistical analysis of these data revealed that serum (CCM)-expanded DPSCs at early passages showed an overall significantly higher expression of STRO-1 (p > 0.001) and SSEA-4 (p > 0.001) but not of SSEA-1 compared to StemMacs and StemPro-expanded DPSCs, with no substantial differences between the latter two. On the other hand, significant medium-related differences were observed in aBMMSC cultures for SSEA-4 (CCM > StemPro, p > 0.001; and StemPro > StemMacs, p > 0.05 in most cell donors) but not for STRO-1 and SSEA-1 expression, the latter two being overall minimally expressed. Finally, when comparing the two cell types, higher SSEA-1 (p > 0.001) and SSEA-4 (p > 0.001) expressions were recorded for DPSC cultures expanded with all three media, as compared to the respective aBMMSC cultures, while substantially higher STRO-1 expression was observed in CCM-expanded DPSCs (15.2 ± 17.7, with, however, very high variability observed among different cell donors, as evidenced by the high SD), compared with all other cell and media groups, where STRO-1 expression was overall quite low (in the range of 2–8%).
Senescence and telomere length analysis
To investigate whether the observed differences in cell growth rates, immunophenotypic profiles and morphological characteristics were associated with replicative cell senescence (as an indication of defective proliferative capacity), the SA-β-gal activity in DPSC and aBMMSC cultures was evaluated after prolonged expansion under serum-based and serum-free conditions (Fig. 4a–c). A significant increase of SA-β-gal-positive cells with increasing cell passage was observed for both cell types (p > 0.001) and all three-expansion media (p > 0.001). It was also shown that the number of SA-β-gal-positive cells was significantly higher for aBMMSCs compared to DPSCs at early (p < 0.001 for CCM, p = 0.0287 for StemMacs and p = 0.0001 for StemPro), middle (p < 0.001 for CCM, p = 0.3957 for StemMacs and p = 0.0087 for StemPro) and late (p < 0.001 for CCM, StemMacs and StemPro) passages. These results were statistically significant with only the exception of StemMacs-expanded cells at the middle passages, where both aBMMSC and DPSC cultures showed similar, relatively high numbers of SA-β-gal-positive cells. Furthermore, DPSCs expanded with StemMacs (at early (p = 0.0122), middle (p < 0.0001) and late (p = 0.0015) passages) and with StemPro (only at late passages, p < 0.0001) demonstrated a significantly higher number of SA-β-gal-positive cells compared to those expanded with the serum-based CCM. In contrast, no major differences were observed for aBMMSCs expanded with the three different media, except for those expanded with StemMacs showing significantly higher numbers of SA-β-gal-positive cells at late passages (p < 0.0001), which confirms growth kinetics/morphological data.
To further evaluate the safety of oral MSC expansion, the telomere length was investigated as an additional measure of MSC aging [29] (Fig. 5a–d). A consistent decrease of telomere length was found in both DPSC and aBMMSC cultures under all culturing conditions. However, the noted telomere shortening was not statistically significant (p ≥ 0.2600) for any of the culture media or types of cells. Telomere length decreased from 11.6 (4.5) to 10.3 (4.2) kbp in CCM-expanded (p = 0.9740), from 12.2 (4.3) to 11.4 (4.2) kbp in StemMacs-expanded (p = 0.9825) and from 13.4 (4.1) to 11.8 (3.6) kbp in StemPro-expanded (p = 0.9329) DPSCs from p.2 to p.10; the respective values for the aBMMSCs were from 12.2 (4.5) to 10.6 (3.5) kbp in CCM-expanded (p = 0.9908), from 14.2 (6.3) to 10.8 (4.4) kbp in StemMacs-expanded (p = 0.9067) and from 16.8 (5.8) to 12.8 (6.1) kbp in StemPro-expanded (p = 0.9940) cells.
Expression of lineage-specific markers
Major differences were observed in lineage-specific gene expression patterns of oral MSCs expanded under serum-based vs serum-free conditions, demonstrating similar trends in both DPSCs and aBMMSCs, despite their differences in their origin. Specifically, both DPSCs and aBMMSCs expanded under serum-free conditions exhibited a significantly higher baseline (at early passages, p.2–3) expression of osteogenic markers (ALP, BMP-2), as compared to serum-expanded cells. Despite interindividual variations, this finding was consistent for most of the cell cultures, except for BMP-2 which was expressed in low levels at baseline by DPSCs expanded with StemPro, similarly to serum-expanded DPSCs. Further, the initially higher baseline expression of ALP in serum-free-expanded cells continued to increase with passaging (or occasionally remained stable), while the initially extremely high expression of BMP-2 significantly decreased (or remained stable in some StemMacs-expanded DPSC donors) (Fig. 6a–d). For StemPro-expanded DPSCs, the initially low BMP-2 expression significantly increased with passaging, unlike for CCM-expanded DPSCs where no significant upregulation of either ALP or BMP-2 generally occurred with prolonged expansion. It should be noted that all of these data were derived from simple passaging without the use of any osteogenic inductive medium.
Similar expression patterns of chondrogenic differentiation markers occurred in long-term expanded DPSCs and aBMMSCs. The chondrogenesis-related transcription factor SOX-9 was substantially upregulated in oral MSCs expanded under both serum-based and serum-free conditions in the majority of cell donors. In contrast, the baseline expression of ACAN gene, encoding the cartilage-specific proteoglycan core protein (CSPCP) or aggrecan, was downregulated in CCM-expanded oral MSCs and was entirely eliminated (no expression) in oral MSCs expanded with both serum-free media (Fig. 6e–h).
In contrast, the expression of PPAR-γ, an adipose tissue-located peroxisome proliferation-activated receptor, and of the adipose-tissue specific lipoprotein lipase (LPL) were in most cases downregulated (in serum-expanded oral MSCs, except PPAR-γ in donor 1) or completely eliminated (in serum-free expanded oral MSCs) after consecutive passaging (Fig. 6i–l).
Taken together, current data indicate that the prolonged expansion of oral MSCs under serum-free, cGMP conditions correlates with overexpression of osteogenesis-related markers, accompanied by complete elimination of adipogenesis and chondrogenesis-related markers, while serum-based expansion causes only minor such changes (i.e., relatively stable levels of osteogenesis-related genes, and moderate downregulation of chondrogenesis-related and adipogenesis-related genes, such as ACAN and LPL, respectively).
Osteogenic and chondrogenic differentiation potential
To clarify whether the substantial differences in gene expression patterns during prolonged expansion also reflect differences of oral MSC differentiation potential, DPSCs and aBMMSCs at early, middle and late passages were induced to differentiate toward osteogenic and chondrogenic lineages. This allowed direct comparisons regarding the lineage-specific differentiation potential toward osteogenic and chondrogenic phenotypes (which are of paramount importance in TE) when different culture systems were used at various passages.
Serum-based medium (CCM)-expanded DPSCs sustained the potential for osteogenic differentiation from early to late passages, indicated by significant, time-dependent upregulation of ALP (p < 0.0001 for all passages), BMP-2 (only for early and middle passages, p < 0.0001) and BGLAP (p < 0.0001 for all passages) up to day 14 following induction. This potential was significantly higher in early-passaged cells but gradually diminished in middle and late-passaged cells, evidenced by the comparative expression of ALP, BMP-2 and BGLAP at day 14 post induction (Fig. 7a, c, e). In contrast, CCM-expanded aBMMSCs enabled upregulation of ALP (p = 0.0098) and BMP-2 (p = 0.0123) (but not BGLAP that was actually significantly downregulated) only at early passages, while this ability diminished at middle and late passages (Fig. 7b, d, f).
StemMacs-expanded DPSCs showed significant downregulation of the extremely high expression of ALP (p < 0.0001 for all passages) and BMP-2 (p < 0.0001 for early and middle; p = 0.0031 for late passages) present at baseline (as described in previous paragraph and Fig. 6), while the initially low expression of the late mineralization marker BGLAP gradually increased (p < 0.0001 for early; p = 0.0075 for middle; nonsignificant for late passages, p = 0.1150) up to 14 days (Fig. 7a, c, e). The ALP and BMP-2 downregulation was subdued in early-passaged compared to middle and late-passaged cells, while the BGLAP upregulation was more pronounced at early compared to late passages.
In StemMacs-expanded aBMMSCs the initial extremely high expression of ALP (p < 0.001 for all passages, except late p = 0.0717) and BGLAP (p < 0.0001 for all passages) declined (Fig. 7b, f), in contrast to BMP-2 which demonstrated a continuously increasing expression, reaching a peak at the middle passages (Fig. 7d). Multiple comparisons between early, middle and late-passage groups showed that BMP-2 upregulation was significantly higher for middle passages (p = 0.0003 compared to early passages at day 14), while BGLAP downregulation was less pronounced at early compared to middle and late passages.
Regarding the StemPro-expanded DPSCs, a significant upregulation of ALP (p < 0.0001 for all passages) as well as of BMP-2 and BGLAP expression was found during osteogenic differentiation (p < 0.0001 for both markers but only at early and/or middle passages) (Fig. 7a, c, e). Multiple comparisons showed that there were no major differences in ALP expression among passages, while BMP-2 and BGLAP expression was more pronounced at early and/or middle passages (Fig. 7a, c, e). Finally, in StemPro-expanded aBMMSCs ALP was upregulated only at early (p = 0.0059) and middle (p = 0.0009) passages, whereas BMP-2 and BGLAP were downregulated or remained stable respectively (Fig. 7b, d, f).
In line with these data, the AR-S-based mineralization assay confirmed that DPSCs expanded with all three-media retained a gradually declining (for CCM and Stem Pro; p < 0.0001) or increasing (for StemMacs; p < 0.0001) mineralization potential after prolonged passaging. However, aBMMSCs showed a higher mineralization potential when expanded under serum-free as compared to serum-based conditions at early passages (p < 0.0001 for comparisons of CCM with both StemMacs and StemPro), while this potential was entirely eliminated for all media at middle and late passages.
A similar analysis for chondrogenic differentiation potential pointed out that CCM-expanded DPSCs demonstrated an increasing chondrogenic differentiation potential with passaging, as signified by increasing expression of ACAN (p = 0.0324, p = 0.0003 and p < 0.0001 at early, middle and late passages, respectively, at day 7 post induction); these effects were more pronounced at late as compared to middle and early passages (Fig. 7i, k). In contrast, CCM-expanded aBMMSCs showed a significant gradual decline in chondrogenic differentiation potential with passaging, as evidenced by diminishing upregulation of ACAN (Fig. 7j, l). For the two remaining media, it was shown that StemMacs-expanded DPSCs and aBMMSCs notably lost their entire chondrogenic potential at all passages, while StemPro-expanded DPSCs and aBMMSCs showed a remaining chondrogenic potential, indicated by the expression of SOX-9 and ACAN that, however, declined with passaging (Fig. 7i–l).