Stemness Maintenance Properties in Human Oral Stem Cells after Long-Term Passage

Background. Neural crest-derived mesenchymal stem cells (MSCs) from human oral tissues possess immunomodulatory and regenerative properties and are emerging as a potential therapeutic tool to treat diverse diseases, such as multiple sclerosis, myocardial infarction, and connective tissue damages. In addition to cell-surface antigens, dental MSCs express embryonic stem cell markers as neural crest cells originate from the ectoderm layer. In vitro passages may eventually modify these embryonic marker expressions and other stemness properties, including proliferation. In the present study, we have investigated the expression of proteins involved in cell proliferation/senescence and embryonic stem cell markers during early (passage 2) and late passages (passage 15) in MSCs obtained from human gingiva, periodontal, and dental pulp tissues. Methods. Cell proliferation assay, beta galactosidase staining, immunocytochemistry, and real-time PCR techniques were applied. Results. Cell proliferation assay showed no difference between early and late passages while senescence markers p16 and p21 were considerably increased in late passage. Embryonic stem cell markers including SKIL, MEIS1, and JARID2 were differentially modulated between P2 and P15 cells. Discussion. Our results suggest that the presence of embryonic and proliferation markers even in late passage may potentially endorse the application of dental-derived MSCs in stem cell therapy-based clinical trials.

Many tissues of the craniofacial region, including the dental pulp and periodontal ligament, are originated from the ectodermal neural crest during embryogenesis [14]. Consequently, MSCs originated from these adult dental tissues express embryonic stem cell markers, such as NANOG, SSEA4, SOX2, and Oct4 [15], along with cell-surface antigens. Expression of embryonic stem cell markers in dental MSCs might be helpful in stem cell therapy-based clinical trials; however, surplus quantity of stem cells is required to accomplish any clinical trials. Thus, multiple subcultures from the initial cells are needed to obtain the optimal cell number for transplantation. Nevertheless, it is important to mention here that prolonged culture of these dental and other adult MSCs can modulate the properties of stemness, including proliferation and differentiation, over time [16][17][18].
In the present study, we have investigated the expression of proteins involved in cell proliferation/senescence and embryonic stem cell markers, including NANOG, SOX2, and Oct4, in hPDLSCs, hDPSCs, and hGMSCs at early passage (passage 2, P2) and late passage (passage 15, P15).

Materials and Methods
2.1. Ethic Statement. The study was approved by the Ethical Committee at the Medical School, "G. d'Annunzio" University, Chieti, Italy (number 266/April 17, 2014). All subjects enrolled in the study signed the informative consent form before tissue collection.
2.2. Cell Culture Establishment. hPDLSCs were isolated from the periodontal tissue and hDPSCs from the dental pulp of noncarious third molars extracted for orthodontic purpose, as previously described [19]. Gingival tissues were collected during surgical gingival resection for the extraction of a supernumerary tooth or for orthodontic reasons. hGMSCs were detached after their spontaneous migration from tissue samples as reported by Soundara Rajan et al. [20]. All donors were in good general health and exempt from oral and systemic diseases. Cells were cultured using MSCGM-CD medium (mesenchymal stem cell growth medium chemically defined) (Lonza, Basel, Switzerland) and were maintained in an incubator at 37°C in a humidified atmosphere of 5% CO 2 in air. Cells were subcultured until P2 and P15. All experiments were performed in triplicate. Cells derived from each donor have been used separately.  [21]. Data were analyzed using FlowJo™ software (TreeStar, Ashland, OR, USA). Mean fluorescence intensity ratio (MFI ratio) was calculated by dividing the MFI of positive events from the MFI of negative events [22].

Cytofluorimetric Evaluation
2.4. Cell Viability Assay. Viability of hPDLSCs, hDPSCs, and hGMSCs at P2 and P15 were determined using the 3-(4,5dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide (MTT) method. 2×10 3 cells/well were placed in a 96-well tissue culture plates and incubated at 37°C for 24, 48, 72 h, and 1 week. At each time point, MTT solution (20 μl) (Promega, Milan, Italy) was added to each well to detect the metabolic activity of the cells. All plates were cultured in the dark for 3 h at 37°C. Supernatants were read at 650 nm wavelength using a microplate reader (Synergy HT, BioTek Instruments, Winooski, VT, USA). The doubling time of Trypan blue harvested cells at 24, 48, 72 h, and 1 week of culture and was calculated by using an algorithm available online (http:// www.doubling-time.com).
2.5. Induction of Mesengenic Differentiation. Human PDLSCs, hDPSCs, and hGMSCs at P2 and P15 were induced to mesengenic differentiation as reported by Trubiani et al. [23]. Briefly, cells were stained with alizarin red S and adipo oil red solution to evaluate osteogenic and adipogenic Germany) was used to analyze stained cells, using a Plan Neofluar oil immersion objective (63×). Micrographs were obtained using excitation lines at 488 nm for argon laser beam and at 543 and 665 nm for a helium-neon source.
CA, USA) was employed to infer biological functions of the selected gene datasets. IPA predicts functional characterization based on known gene functional interactions and ranks them by a significance score [27]. p16 and p21 gene expressions were analyzed by qRT-PCR using the same cDNA employed for expression arrays. Specific primer and probe sets employed were purchased from ThermoFisher Scientific (Waltham, MA, USA): p16 Hs00923894_m1 and p21 Hs01040810_m1. qRT-PCR was performed in a total volume of 30 μl containing KAPA Probe Fast Abi Prism qPCR Kit (KAPA Biosystems), 25 ng of cDNA, and 1 μl of primer-probe mixture (20x) on an Abi 7900HT Sequencing Detection System. The selected gene relative expression was corrected against GAPDH (Hs02758991) used as endogenous control (ThermoFisher Scientific, Waltham, MA, USA). Real-time amplification conditions were as follows: 10 minutes at 95°C followed by 40 cycles of 15 seconds at 95°C, and 1 minute at 60°C. Each sample was run as triplicate. The ΔΔCt method was used to compare relative fold changes between samples and control. t-test was used to assess the p value, considering data significant when p < 0 05.

Statistical
Analysis. Data were analyzed using GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA, USA) with oneway ANOVA test, followed by a Bonferroni post hoc test for multiple comparisons. A p value < 0.05 was considered statistically significant.
MTT data were confirmed by Trypan blue exclusion test staining. The results from Trypan blue staining of hPDLSCs, hDPSCs, and hGMSCs at P2 and P15 displayed the logarithmic growth during the culture time (Figures 1(b), 1(d), and 1(f), resp.).

p16 and p21
Analysis. p16 and p21 showed a slightly increased expression pattern at P15 when compared to P2 in all three primary cultures (p < 0 05) (Figures 4(a), 4(b), and 4(c)). According to RT-PCR results, western blot showed an upregulation of both senescence markers examined in hPDLSCs, hDPSCs, and hGMSCs at P15. In particular, a low expression of p16 and p21 was noticed at P2 in all primary cultures (Figures 4(d), 4(e), and 4(f)). Densitometric analysis of specific bands confirmed p16 and p21 expressions in hPDLSCs, hDPSCs, and hGMSCs (p < 0 05) (Figures 4(g), 4(h), and 4(i), resp.). β-Actin was used as a loading control protein.   Figure 7: Gene modulation of hDPLSCs. IPA-inferred cluster analysis of the hPDLSC dataset of significantly expressed genes at P15. transcripts (GBX2, JARID2, and SKIL). IPA functional analysis of the three gene datasets revealed that these genes are mainly involved in cellular, embryonic, and tissue development and in gene expression regulation ( Figure 6). IPA analysis revealed that hGMSCs and hPDLSCs have an increase in differentiation process, in particular ectodermal differentiation whereas hDPSCs showed a reduction in transcriptional activity and a lower differentiation pattern, although overexpressing JARID2 and SKIL which are involved in osteo-and muscular-differentiation patterns (data not shown). IPA-inferred gene network analysis of the three gene datasets demonstrated that at P15 the downregulation of SOX2 and POU5F1 triggers the modulation of the analyzed transcripts in a different manner for the three conditions (Figures 7, 8, and 9). In fact, the intracellular pathways were differentially regulated, showing the specific activation of HESX1, MEIS1, and TCF7L1 in hPDLSCs; the activation of CDYL, GATA6, SALL1, and SMARCAD1 in hGMSCs; and the activation of GBX2, JARID2, and SKIL in hDPSCs. IPA functional analysis demonstrated that this differential gene expression modulation is due to the ability of these cells to differentiate in specific lineages.

Discussion
Research in recent years attested that dental tissues serve as an alternative source for MSCs and regenerative application of dental MSCs has been demonstrated not only in dental regeneration but also in other diseases [28]. Together with stem cell-associated cell surface markers, owing to their neural crest origin, dental MSCs express embryonic stemness markers as well [29], which may influence them to differentiate into diverse cell lineages in vitro.
MSCs from oral tissues can be considered an interesting accessible autologous platform of stem cells, providing an alternative source for regenerative medicine. In particular, dental pulp and periodontal ligament express proteins similar to BMSCs and can be induced toward osteogenic, adipogenic, chondrogenic, and neurogenic differentiation [26,30]. Large-scale expansion of MSCs with low grade of senescence is very crucial for stem cell transplantation. However, continuous passages of adult MSCs for a longer period may affect the embryonic stemness properties, including proliferation and differentiation markers [17,18].
In the present study, we have investigated the expression of embryonic markers and proteins involved in proliferation/ senescence in hPDLSCs, hDPSCs, and hGMSCs at two different passages: P2 and P15. All oral primary cultures express surface antigen markers linked with MSCs, such as CD13, CD29, CD44, CD73, CD90, CD105, CD166, HLA-ABC, NANOG, OCT4, SSEA4, and SOX2. In addition, we noticed that the degree of cell proliferation at P2 and P15 remain unchanged among hPDLSCs, hDPSCs, and hGMSCs. These results suggest that these dental MSCs are highly proliferative even at P15. Cell proliferation efficacy of hPDLSCs, hDPSCs, and dental periapical follicle MSCs has been studied previously where it has been reported that hDPSCs showed adequate proliferation between passages 11-14 [31] and that periapical follicle MSCs displayed greater cell proliferation than hDPSCs and hPDLSCs [32]. However, in our study, we did not find any difference in the cell proliferation rate in hPDLSCs, hDPSCs, and hGMSCs at both P2 and P15.
We then analyzed the expression of senescence markers in hPDLSCs, hDPSCs, and hGMSCs. Continuous passages may likely induce senescence in MSCs and that cellular senescence contributes to aging and age-related diseases [33], which possess danger to transplant long-term-cultured MSCs for stem cell therapy. Senescence-associated β-galactosidase staining showed less positivity at only P15, while no staining was noticed at P2 in all the dental MSCs. On the other hand, senescence marker p16 expression was slightly increased at  Figure 9: Gene modulation of hGMSCs. IPA-inferred cluster analysis of the hGMSC dataset of significantly expressed genes at P15. the 15th passage in hPDLSCs and hDPSCs and not in hGMSCs. Meanwhile, p21, another senescence marker, remained unchanged at both P2 and P15 in hPDLSCs, hDPSCs, and hGMSCs. Additional experiments with more numbers of passages need to be performed to study senescence phenomena in these dental MSCs. Lastly, we studied the expression of selective embryonic stemness markers at early and late passages. RT-PCR technique revealed that embryonic markers OCT4, SSEA4, SOX2, and NANOG were present at P2 of hPDLSCs, hDPSCs, and hGMSCs. Other embryonic markers SALL1 and OTX1 were absent in P15 of hPDLSCs. NANOG expression was diminished in P15 of hPDLSCs and hDPSCs, while it remained unaltered in hGMSCs. In addition, we noticed heterogeneous expression of other embryonic markers such as CDYL, FOXC1, HESX1, JARID2, MEIS1, and MYST3 at P15 in hPDLSCs, hDPSCs, and hGMSCs. A varied expression of embryonic stemness markers in MSCs obtained from the human bone marrow, adipose tissue, heart, and dermis at early passages has been already reported previously [34]. Although derived from neural crest tissues, results from our data demonstrated that MSCs of different dental tissues might acquire different changes in markers associated with the stemness properties over prolonged culture expansion.
Neurogenic markers such as NEUROG1, REST, and ZFHX3 were differentially modulated among hPDLSCs, hDPSCs, and hGMSCs at P15, which assume that continuous subculture of these dental MSCs may preferably channel them to differentiate into neural progenitor cells. Considering the neural crest origin of dental MSCs, additional experiments must be performed to investigate the potential of dental tissue-derived MSCs on autonomous neurogenesis differentiation. Expression of markers involved in the regulation of cell growth, proliferation, and DNA repair mechanism such as RIF1 and SET was downregulated, while SKIL expression was upregulated at P15 in hPDLSCs, hDPSCs, and hGMSCs. Other cell proliferation markers SMARCAD1, TCFL1, and TRIM24 were differentially modulated at P15. These results corroborate our cell proliferation data, suggesting marked rate of proliferation at P15 in hPDLSCs, hDPSCs, and hGMSCs.

Conclusion
In summary, our results demonstrated that hPDLSCs, hDPSCs, and hGMSCs do proliferate at a similar rate in P2 and P15. Senescence marker p16 was only slightly modified in the late passage, while p21 remained unchanged. Embryonic stemness markers were differentially modulated in P15. We conclude that expression of embryonic and proliferation markers at late passage with mild regulation of senescence may potentially recommend the use of oral-derived MSCs in stem cell therapy.

Conflicts of Interest
The authors declare that there is no conflict of interest regarding the publication of this paper.

Authors' Contributions
Francesca Diomede and Thangavelu Soundara Rajan contributed equally to this work. Emanuela Mazzon and Oriana Trubiani contributed equally to this work as senior authors.