Effect of Long-Term Cryopreservation on the Stemness of Stem Cells of Apical Papilla

Stem cells of apical papilla (SCAPs) are considered a subpopulation of dental stem cells with unique properties. They originate from a developing tissue, the apical papilla of developing teeth, a characteristic that enhances their stemness. Banking of these stem cells can offer a source of dental stem cells for future regenerative therapies. Until now, only the effect of six months' cryopreservation on SCAPs has been studied. In this study, the long-term (19 months) effect of cryopreservation on SCAPs was examined by means of estimation of their differentiation's capacity, flow cytometry immunophenotypical characterization, and molecular characterization of the main transcriptional factors that coincide with pluripotency. As was indicated from our results, 19-month cryopreservation of SCAPs did not affect negatively their stemness; since no significant difference was observed on their typical fibroblast-like morphology, they retained their differentiation capacity, and no discrepancies were found either on immunophenotypical level or molecular level.


Introduction
Regenerative endodontic procedures (REPs) are based on the use of the "holly triad," stem cells (SCs), growth factors, and scafolds [1]. Apical papilla comprises the main source of stem cells that are employed during REP, since, in most of these cases, dental pulp is necrotic, which means absence of dental pulp stem cells.
Stem cells of apical papilla (SCAPs) are considered a subpopulation of dental stem cells with unique properties [2]. Te most striking characteristic of SCAPs is their origin, as they derive from a developing tissue, the apical papilla of the developing tooth. On the other hand, the rest of stem cells in the human body (somatic stem cells) originate from fully developed organs. Te above characteristic of SCAPs enhances their stemness.
Tey are present only during the early stages of tooth development, since their residence, apical papilla, is resolved after the fulfllment of root development [3]. In that line, banking of SCAPs can ofer a source of dental stem cells for future regenerative therapies. Until now, only the efect of six months' cryopreservation on SCAPs has been studied [4].
Terefore, in this study, the long-term efect of cryopreservation will be examined by means of estimation of their diferentiation's capacity, fow cytometry immunophenotypical characterization, and molecular characterization of transcriptional factors of genes that coincide with pluripotency before and after 19 months cryopreservation.
alter the gene expression of the transcription factors. Prior to extraction, radiographic examination was performed for the verifcation of the presence of the apical papilla. Te extractions were held under the most aseptic conditions and most gentle atraumatic manipulations for the preservation of apical papilla. Te teeth were proceeded immediately for apical papillae' separation.
2.1. SCAP Isolation, Culture, and Proliferation. SCAPs were isolated and cultured as previously described by Sonoyama et al. [2]. Briefy, apical papilla was digested in a solution of 3 mg/ml collagenase type I and 4 mg/ml disposed for 30 min at 37°C, passed through a 70 mm strainer to obtain a singlecell suspension, and seeded into 25 cm 2 culture fasks containing alpha-modifcation of Eagle's medium supplemented with 15% fetal bovine serum, ascorbic acid 2-phosphate, glutamine penicillin, and streptomycin. Cultures were incubated at 37°C in a humidifed atmosphere supplemented with 5% CO 2 . Medium was changed twice a week. Upon reaching 80% confuence, SCAPs were detached using 0.05% Trypsin-EDTA, reseeded, and cultured for further study.

2.2.
Cryopreservation. Between 2-3 passage SCAPs were detached using 0.05% Trypsin-EDTA, centrifuged at 800g for 10 min, and resuspended in fetal bovine serum containing 10% DMSO in a cellular density of 3-4 * 10 6 cells per ml. Gradual freezing was performed, and the cells were fnally stored in liquid nitrogen for 3, 8, and 19 months to test their characteristics upon thawing.

Before and After Cryopreservation Periods in Diferent
Time Points (3,8 Table 2) as well as HPLC Purifcation (Lab Supplies, Athens, Greece), Q-PCR for 10 ng of RNA template was performed, the results of which were analyzed by using ddCt algorithm for the analysis of the relative changes in gene expression. Rotor Gene 6000 genetic amplifcation detection system (Corbett Life Science) was used to perform the reaction. GAPDH was used as house-keeping gene for the normalization of gene expression.

Statistical
Analysis. Data were presented as mean ± SD, and Student's t-test (unpaired, two-tailed) was used for the two-group comparisons. Diferences were considered statistically signifcant at a value of p ≤ 0.05.

Before Cryopreservation.
SCAPs were isolated according to the Sonoyama et al. [2] method. First attachment of the stem cells on plastic surfaces was observed 1 to 4 days after cells' plating. SCAPs showed the typical fbroblast-like morphology after attachment while their expansion rate was gradually augmented ( Figure 1). Te cultures were ready for further manipulation between the third and fourth week of incubation (3.5 × 10 6 cells). Te induced diferentiation of SCAPs confrmed osteogenic, adipogenic, and chondrogenic capacity as confrmed with positive Alizarin Red, Oil Red, and Alcian blue staining, respectively, and in comparison, with the control unstained groups ( Figure 2).
In the transcriptional level, as proved by Q-PCR, most transcription factor-related genes are highly expressed especially in case of NANOG, OCT4, ESRRB, and ZNF878 genes where 14, 11, 17, and 12-fold augmentation was observed ( Figure 4).

After Cryopreservation.
After 3, 8, and 19 months of cryopreservation and thawing of SCAPs, their frst attachment on plastic surfaces was observed 2 to 6 days after cells' plating. SCAPs retained the typical fbroblast-like morphology after attachment while their expansion rate was gradually augmented as in the primary cultures ( Figure 5). Te cultures were ready for further manipulation between the third and fourth week of incubation (3.5 × 10 6 cells).
SCAPs retained their diferentiation capacity after 3, 8, and 19 months of cryopreservation as it was confrmed by induced diferentiation of SCAPs to osteogenic, adipogenic, and chondrogenic lineage positive staining with Alizarin Red, Oil Red, and Alcian blue, respectively, and in comparison, with the control unstained groups (data not shown).

Gene
Main properties NANOG (i) Regulation of the diferentiation and the pluripotency (ii) Sustaining the pluripotency of epiblast and impeding it from diferentiation (iii) Induction of self-renewal and the preservation of pluripotency (5) OCT4 (i) Yamanaka factor, essential for the generation of pluripotent stem cells of fbroblasts (ii) Regulation of the pluripotency and diferentiation of ESC (6) CMYC (i) Yamanaka factor, essential for the generation of pluripotent stem cells of fbroblasts (6) (ii) Regulation of the pluripotency and diferentiation of ESC (6) (iii) Regulation of cell cycle entry and apoptosis (7) (iv) Promotion of cell growth proliferation (7) (v) Suppression of terminal diferentiation (7) SOX2 (i) Yamanaka factor, essential for the generation of pluripotent stem cells of fbroblasts ( Figure 3). Furthermore, cryopreservation did not afect the SCAPs in transcriptional level, as depicted by Q-PCR: the majority of transcription factor-related genes were highly expressed in SCAPs after 3, 8, and 19 months of cryopreservation; also, NANOG, OCT4, ESRRB, and ZNF878 genes maintained their high augmentation (Figure 4).
Te above results indicate that 19-month cryopreservation did not afect negatively the stemness of SCAPs; since no signifcant diference was observed on their typical fbroblast-like morphology, they retained their diferentiation capacity, and no discrepancies were found either on immunophenotypical level or molecular level.

Discussion
Stem cells of apical papilla (SCAPs) have recently gained great attention due to their implication in regenerative endodontic procedures (REPs). Tey are considered the "apical papilla treasure" [17]. Along with dental pulp stem cells (DPSCs) (18), they consist of the most representative dental stem cells. Nevertheless, their superiority over DPSCs is well established [17,18]. SCAPs exhibit faster colonyforming and greater deposition of mineralized tissue matrix than DPSC [19]. Furthermore, SCAPs can survive the pulpal and periapical infammation, due to the collateral vascularization of the apical papilla [20], in contrast to DPSC, that are obscured in necrotic dental pulps, and thus, unobtainable for REPs. Tey, also, appear the ability of proliferation in large numbers in vitro [21].
Te immunophenotypical characterization by FCM has shown that the isolated primary cell populations comprised the characteristics of SCAPs. Pluripotency marker CD24 is considered SCAP specifc, since it cannot be detected in other mesenchymal stem cells (MSCs), including DPSCs [22]. In this study, the cell populations were highly positive for CD24 (expression: 98.70%). According to Liu et al. [22], to evaluate the pluripotency, high positivity to CD24 is a perquisite for the selection of cell populations. Tey were, also, highly positive for CD146 (97.44%) that indicates their perivascular location [22] as well as highly positive for the typical MSC markers CD90 (99.2%), CD105 (98.65%), CD29 (94.20%), and CD44 (92.00%), indicating their mesenchymal origin [22]. Finally, they were negative for the leukocyte precursor marker CD45 (0.79%) and hematopoietic marker CD34 (0.94%). Tis almost lack of expression for CD45 and CD34 confrms frstly their stromal origin and secondly the purity of cell cultures, without contamination with hematopoietic precursors [2,23].
Te abovementioned immunophenotypic characteristics of SCPAs were preserved after 19 months cryopreservation, as it was shown from our results, indicating safe cryopreservation of SCAPs.
Te morphological diferentiation of SCAPS to osteocytes, adipocytes, and chondrocytes before and after cryopreservation also confrms that cryopreservation did not afect negatively their stemness capacity and ability to be diferentiated towards the three germ layers successfully, as it was shown after the appropriate staining of induced differentiated cultures.
In this study, the expression of genes that coincide with pluripotency has been evaluated in SCAPs for the frst time, before and after cryopreservation, to characterize in detail the molecular profle of this cell subpopulation and study the efect of cryopreservation on their molecular profle.
From our results, SCAPs express in diferent x fold amount NANOG, OCT4, C-MYC, SOX2, KLF4, SALL4, ESRRB, and ZNF217 genes responsible for diferentiation and reprogramming of cells which are commonly expressed at high levels in embryonic (ESC) and mesenchymal stem cells (MSC). Although most transcription factor-related genes, like NANOG, OCT4, and ZNF878, are highly expressed, the ESSRB gene expression is remarkably high reaching 17 x-fold augmentation.
ESSRB is a crucial determinant for the maintenance of pluripotency since its depletion or removal leads to diferentiation. Regarding self-renewal, ESSRB can functionally substitute NANOG: in the absence of GSK3I and LIF, NANOG cannot preserve self-renewal while ESSRB has this capacity [11]. Although ESSRB can rescue self-renewal in the absence of NANOG, with simultaneous presence, they can express enhanced efciency on self-renewal. Synergetic action of ESSRB with NANOG induces the pluripotency of  International Journal of Dentistry diferentiated cells and stabilizes ESC self-renewal through positive feedback [12]. Additionally, ESSRB promotes reprogramming of somatic cells to pluripotent state, even in the absence of NANOG [12], and has the unique capacity to reprogram NANOG -/cells to naïve pluripotency. ESSRB is considered as a marker of reprogramming progression in somatic cells and along with Nanog can substitute SOX2 while OCT4 remains needful for vigorous reprogramming [13].
ESSRB expression provides stability to the network of naïve pluripotency, and ESSRB positive cells are considered to represent an "elite" subpopulation [13]. Terefore, SCAPs, as indicated from our results, can be numbered among the "elite." Human pluripotent stem cells in naïve state highly express, among other genes, ESSRB, NANOG, OCT4, SOX2, and KLF4 [14]. As shown from our results, SCAPs highly express Essrb, Nanog, Oct4 and in a lower grade SOX2 and KLF4, so it could be considered "naïve-like" stem cells. Te overexpression of ESRRB gene in SCAPs, along with the high expression of NANOG, OCT4, and ZNF878 genes, as found in our study, confrms their undiferentiated state and rationalizes their high pluripotency potentiality. Additionally, to their capability for dentinogenic diferentiation [2], SCAPs exhibit high osteogenic potential [19].
Our results are in agreement with the results of Bakopoulou et al. [28,29] and Wu et al. [30] regarding the expression of NANOG, and OCT4, while regarding the rest of the genes examined in this study, no other data exist for their expression in SCAPs.
Furthermore, it also evident by this study that cryopreservation did not afect the molecular profle of SCAPs, since they continued to express the abovementioned genes in almost the same levels, as in primary cultures.
SCAP-based regeneration dental therapies include regenerative endodontic procedures whose main goal is the fulfllment of root development in immature permanent necrotic teeth and cell-based therapies in combination with minimal access fap surgery for periodontal reconstruction [31][32][33][34]. Besides dentin-pulp and periodontal regeneration, SCAP-based therapeutic applications are implicated in bioroot engineering, in the regeneration and repair of neural tissue, and in ischemic diseases even in immunotherapies [35]. New data in the translational research regarding SCAPs are shown in our study that further elucidate and rationalize their pluripotency capacity.
Our results, on the study of cryopreservation of SCAPs, agree with the results of Ding et al. [4], where six-month freezing was studied.

Conclusions
Stem cells of apical papilla (SCAPs) are obtained from extracted human immature third molars or immature teeth extracted for orthodontic purposes during routine dental procedures, which are less invasive compared with the harvesting processes required for other types of stem cells, e.g., bone marrow stem cells. Tis high accessibility, in combination with their multipotential diferentiation capacity along with their undiferentiated state, and their ability for long-term safe cryopreservation and banking, as indicated from our results, highlight SCAPs as excellent candidates for regeneration procedures in general. Further studies, with longer banking periods are necessary, to study the efect of cryopreservation on SCAPs.

Data Availability
Te data supporting the conclusions of the study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that there are no conficts of interest regarding the publication of this article.