Cyclin-Dependent Kinase 1 Inhibition Potentiates the Proliferation of Tonsil-Derived Mesenchymal Stem Cells by Delaying Cellular Senescence

Mesenchymal stem cells (MSCs) have been widely used in tissue regeneration and stem cell therapy and are currently being tested in numerous clinical trials. Senescence-related changes in MSC properties have attracted considerable attention. Senescent MSCs exhibit a compromised potential for proliferation; senescence acts as a stress response that prevents the proliferation of dysfunctional cells by inducing an irreversible cell cycle arrest. Here, we established a senescent MSC model using senescence-associated β-galactosidase, proliferation, and cell cycle assays. We further identified novel biomarker candidates for old, senescent tonsil-derived MSCs (TMSCs) using transcriptomics. A plot of the cellular senescence pathway showed cyclin-dependent kinase 1 (CDK1; +8-fold) and CDK2 (+2-fold), and transforming growth factor beta 2 (TGFB2; +2-fold) showed significantly higher expression in old TMSCs than in young TMSCs. The CDK family was shown to be related to cell cycle and proliferation, as confirmed by quantitative RT-PCR. As replicative senescence of TMSCs, the gene and protein expression of CDK1 was significantly increased, which was further validated by inhibiting CDK1 using an inhibitor and siRNA. Taken together, we suggest that the CDK1 can be used as a selective senescence biomarker of MSCs and broaden the research criteria for senescent mechanisms.


Background
Cellular senescence-related changes in mesenchymal stem cell (MSC) properties have attracted considerable attention in the tissue engineering and regenerative medicine fields [1,2]. Senescent MSCs exhibit a compromised potential for proliferation, homing effective migration, tissue differentiation, angiogenesis, and regeneration [3][4][5][6]. However, therapies utilizing MSCs often require ex vivo expansion to generate large quantities of cells required for patients [7][8][9]. Despite their therapeutic potential and safety, MSCs must be serially passaged for an extended period to obtain a sufficient number of cells for in vivo or clinical application [10].
Long-term replicative subculture of MSCs induces continuous changes in cell properties, including decreased proliferation rate, self-renewal, and regenerative potential, resulting in cell cycle arrest and changes in morphology such as cell widening and flattened shape [8,11,12]. Cellular senescence occurs in response to DNA damage, oncogene mutations, oxidative stress, damage-associated molecular pattern molecules from diseased tissue, and various other cellular insults [13][14][15]. In particular, senescence has been well characterized as a stress response that prevents the proliferation of dysfunctional cells by inducing irreversible cell cycle arrest [16,17].
During long-term replicative cell culture, MSCs may undergo molecular changes that result in the acquisition of senescent phenotypes independent of MSC isolation and culture conditions [18]. To achieve a successful regenerative outcome, an increasing number of studies have recently highlighted the roles of cell cycle-related genes or proteins in therapeutic MSCs because they act as key contributors during proliferation largely by regulating cell cycle responses [16]. Additionally, optimized strategies need to be developed to elucidate the cell cycle regulating factors in senescent MSCs for better outcomes in stem cell therapy [19].
Recently, tonsil-derived MSCs (TMSCs) obtained from human tonsil tissues, generally isolated from waste tissues after a tonsillectomy, have been applied as MSC sources for clinical applications owing to the noninvasive nature of tissue collection without unnecessary surgeries and high proliferation rate [20]. Regardless of their source, replicative subculture also leads to a decrease in TMSC functions by leading them to a state of replicative senescence (i.e., in vitro culture aging, also known as the Hayflick limit) [21].
Furthermore, MSCs undergo senescence after a certain number of cell expansion passages in vitro and finally stop the proliferation [22]. Senescent MSCs often exhibit persistent activation of the DNA damage response with concomitant increases in cyclin-dependent kinase (CDK) inhibitors [23]. CDKs regulate various proteins necessary for cell cycle progression and prevent advancement when bound to their cyclin partners [24]. The CDK inhibitors p16INK4a (encoded by CDKN2A) and p21 (encoded by CDKN1A) have been highly implicated in senescence, along with the p53 signaling pathway [25,26]. Specifically, p16 inhibits G1 to S phase transition via inhibition of CDK4 and CDK6. The p21 acts similarly via inhibition of CDK2, among other CDKs [27]. Therefore, it is necessary to understand the association of CDK with the cell cycle to promote the proliferation of senescent MSCs.
In this study, we established a new in vitro senescent stem cell model using various experiments, including the senescence-associated β-galactosidase (SA-β-gal) assay, proliferation assay, and cell cycle assay. Additionally, we investigated differential gene expression patterns between young and old TMSC groups, distinguished by long-term replicative cultivation. Using transcriptomic approaches, we identified novel biomarker candidates for detecting old, senescent TMSCs, thereby elucidating the role of identified biomarkers in the functional recovery of old TMSCs.

Materials and Methods
2.1. Isolation and Culture of TMSCs. TMSCs were isolated from excised palatine tonsillar tissues obtained from four patients following tonsillectomy, as described previously [28]. Tonsillar tissues used in this study were acquired from four donors (2 girls and 2 boys) who had undergone a tonsillectomy due to tonsil hyperplasia without tonsillitis. In addition, the donors consisted of patients under 10 years of age (Supplementary Table 1). Before surgery, all subjects provided their informed written consent for the use of their tissue specimens for research purposes and TMSC isolation according to the guidelines of the Chungbuk National University Medical Center (IRB No. CBNU-202106-BRHR-0073). Tonsillar tissues used in this experiment were collected from patients who underwent tonsillectomy due to tonsil hyperplasia.
The isolated tonsils were mechanically digested by cutting, and the tissues were then chemically digested using collagenase type I (Thermo Fisher Scientific, Waltham, MA, USA) and DNase (Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 30 min. The obtained cell suspension was filtered through a wire mesh, and mononuclear cells were isolated using Ficoll-Paque (GE Healthcare, Piscataway, NJ, USA) density gradient centrifugation. The resulting mononuclear cells were cultured in DMEM-HG (Welgene Inc., Gyeongsan, Korea) supplemented with 10% fetal bovine serum (FBS: certified, US origin, Gibco, Grand Island, NY, USA), 1% antibiotics/antimycotics (A/A), and penicillin/streptomycin (P/S) (Gibco) at 37°C in a humidified 5% CO 2 incubator.
Cells were allowed to adhere to culture plates for 24 h, and adherent mononuclear cells were used as TMSCs. The cells were cultured in DMEM supplemented with 10% FBS, and the medium was changed every two days. When the TMSCs reached 80-90% confluence, they were treated with 0.25% trypsin-EDTA (Gibco) for 3 min. The detached cells were washed twice with PBS and collected by centrifugation at 3,000 rpm for 5 min (Eppendorf, Hamburg, Germany).

Experimental Groups of TMSCs.
After reaching 80% confluence, TMSCs were subcultured onto individual dishes and replicative subcultured to passages 15 to 20 to induce cellular replicative senescence. The maximum number of passages was determined by the proliferative capacity. The TMSCs used in this study were between passages 5 and 20. The growth medium was changed every three days. The in vitro-cultured TMSCs were divided into two groups as follows: young (passages 5-8) and old (passages 15-20) TMSCs.

2.3.
Senescence-Associated-β-Gal Assay. Morphological changes associated with experimental treatments, including increased cell size, altered overall morphology, and decreased proliferative capacity, were assessed with an inverted microscope (Olympus). Senescent TMSCs were detected by SA-β-gal staining using a commercial staining kit (Cell Signaling Technology, Boston, MA, USA) according to the manufacturer's instructions. Briefly, TMSCs were fixed with 4% paraformaldehyde (Biosesang, Seongnam, Korea) for 15 min at 37°C, and the fixed cells were incubated with β-gal staining solution at 37°C overnight in a dry incubator without CO 2 supply. Old cells were identified by their β-gal blue-stained cells under a standard light microscope. Stained cells were expressed as a percentage of total TMSCs.

Cell Cycle and Proliferation
Analysis. The cell cycle stage was examined by measuring the DNA content of nuclei labeled with propidium iodide (PI, Abcam). For the cell cycle analysis, either young or old TMSCs (1 × 10 5 cells) were harvested by trypsinization and gently pelleted by centrifugation at 300 × g for 5 min. The pellets were washed and resuspended in 200 μl DPBS. The resuspended cells were transferred dropwise into 70% ethanol (800 μl) and fixed for 1 week. The fixed cells were collected, washed, and resuspended in PI staining solution (50 mg/ml) containing RNase A (100 mg/ml) and incubated in the dark for 30 min at room temperature. A FACScan (FACSCalibur-S System; BD Biosciences) was used to analyze the cell cycle, and the FlowJo software was used for data analysis. were exported to analyze differentially expressed genes (DEGs). The false discovery rate was controlled by adjusting the p value using the Benjamini-Hochberg algorithm. For a given DEG set, hierarchical cluster analysis was performed using the complete linkage and Euclidean distance as a measure of similarity. Gene enrichment and functional annotation analyses of significant probe lists were performed using the GO and KEGG databases. The statistical significance of the expression data was determined using a local-pooled-error test and measurements of fold change (fc), with the null hypothesis that no difference exists between groups. All data analyses and visualization of DEGs were conducted using R 3.0.2.

CDK1 Knockdown Experiments
Using Inhibitor and siRNA Transfection. CDK1 protein expression in old TMSCs was knocked down using an inhibitor and siRNA. Two days before treatment, the old TMSCs were grown in 6-well plates containing DMEM supplemented with 10% FBS. The CDK1/2 inhibitor III (Enzo Life Sciences, Inc., NY, USA) was used at the 10 μM concentration in DMSO, which is sufficient to induce robust checkpoint arrest in cells at the cell cycle stage. CDK1 siRNA and scrambled siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz). The scrambled control siRNA or CDK1 siRNA was transfected into the old TMSCs using Lipofectamine 2000 and Lipo-PLUS (Thermo Fisher). After the transfection, the cell lysates were collected and analyzed using quantitative RT-PCR analysis.
2.11. Quantitative RT-PCR. TMSCs were plated onto 100 mm 2 culture dishes and incubated at 37°C under 5% CO 2 till they reached 80-90% confluence. Total RNA from TMSCs was extracted using the TRIzol reagent according to the manufacturer's instructions (Life Technologies, Darmstadt, Germany). Isolated RNA was treated with DNase I (Thermo Scientific, Schwerte, Germany) to remove possible genomic DNA contamination and used for cDNA synthesis using Superscript III Transcriptase (Life Technologies) and random hexamer primers (Thermo Scientific) at 50°C for 1 h. Each sample was run in triplicate. Quantitative RT-PCR was conducted using POWER SYBR Green qPCR Master Mix (Life Technologies) and 0.2 μM primer. The primer sequences used were as follows: (1)

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Foster City, CA) under the following cycling conditions: initial denaturation at 95°C for 5 min, 40 denaturation cycles of 30 s at 95°C, and annealing for 40 s at 60°C and 30 s at 72°C, followed by fluorescence measurements.
2.12. Western Blot Analysis. TMSCs were lysed with a RIPA buffer containing protease inhibitor mixture (Roche Applied Science). Equal amounts of protein were loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were transferred onto nitrocellulose membranes with 0.2 μm pore size (GE Healthcare). The membranes were then blocked in TBST with 5% skim milk, and the blots were incubated with appropriate primary antibodies followed by the corresponding secondary antibodies tagged with HRP, which were then visualized using enhanced chemiluminescence reagents. Primary antibodies against the following proteins were used in this study: CDK1, CDK2, cyclin B1, cyclin E2 (Cell Signaling Technology), and GAPDH (AB Frontier). All protein band images from western blot analyses were quantified densitometrically using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
2.13. Statistical Analysis. All data are presented as the mean ± standard deviation (SD). All data graphs were generated using the GraphPad Prism 7 software (GraphPad Software Inc., La Jolla, CA, USA). The significance of differences between the two experimental groups (young and old TMSCs) was analyzed using Student's t-test. Statistical significance was set at p value < 0.05 ( * p < 0:05, * * p < 0:01, or * * * p < 0:001).

Changes of Surface Marker Expressions with Senescence.
The phenotypic characteristics of old TMSCs were assessed     Stem Cells International using representative stem cell surface markers, including hematopoietic cell markers (CD14, CD34, and CD45) and primitive cell markers (CD73, CD90, and CD105) ( Figure 2). Using fluorescence-activated cell sorting (FACS), we found no significant changes in the expression of either hematopoietic or primitive surface protein markers between young and old TMSCs. Young (orange peak) and old TMSCs (blue peak) resulted in completely overlapped peaks, indicating that senescent old cells cannot be distinguished from young cells by using these surface markers.

Transcriptomic Profiles of Cellular Senescence in Young and Old TMSCs.
We performed a transcriptomic analysis of young and old TMSCs. Senescence-related transcriptomic differences were presented as a log2 fold change heatmap distribution (jlog 2 ðfold − changeÞj >2) in Figure 3(a). A complete list of heatmap genes is provided in Table 1. The quality of the DNA-seq data was confirmed by a repeatability check using Pearson's correlation coefficient analysis (p < 0:05). Based on multidimensional scaling (MDS) scores, the old group was distinguishable from the young group (Figure 3(b)). Unsupervised hierarchical clustering showed a clear distinction between the young and old TMSCs (Figure 3(c)).

Molecular Functional Gene Ontology of Highly Regulated
Genes and KEGG Pathway Analysis in Old TMSCs. We categorized differentially expressed genes in old TMSCs according to the molecular function-related gene ontology (GO) classification (MF; Figure 3(d)). The top 20 enriched molecular function (MF) terms were protein heterodimerization activity, ATPase activity, tubulin binding, catalytic activity acting on DNA, microtubule binding, helicase activity, single-stranded DNA binding, DNA helicase activity, extracellular matrix structural constituent, histone deacetylase binding, microtubule motor activity, DNA replication origin binding, DNA secondary structure binding, DNAdependent ATPase activity, single-stranded DNA helicase activity, cyclin-dependent protein kinase regulator, fourway junction DNA binding, histone kinase activity, flap endonuclease activity, and 3′-5′ DNA helicase activity.
To evaluate signaling pathways associated with enriched genes in replicative senescence of old TMSCs, we performed a KEGG pathway analysis (Figure 3(e) Table 2).
Among the three pathways, we focused on the cellular senescence KEGG pathway (Supplementary Figure 1). The identified genes related to cellular senescence and proliferation from the in silico analyses were CDK1, CDK2, and transforming growth factor beta 2 (TGFB2), all of which indicate representative cellular senescence-specific pathways. A plot of cellular senescence KEGG pathway showed that CDK1 (+8-fold change, fc), CDK2 (+2-fc), and TGFB2 (+2-fc) were significantly higher in old TMSCs than in young TMSCs. The gene that presented the most significant changes in expression with respect to senescence (>8-fc) was CDK1, shown to be related to the cell cycle and cell proliferation (Supplementary Table 2).
3.6. Changes of Cell Cycle-Specific Protein Expression and CDK1 Activity in TMSCs. As the replicative senescence of TMSCs progressed, the changes in cell cycle-specific marker protein were examined by western blot analysis of CDK1, CDK2, cyclin B1, and cyclin E2 ( Figure 5). During replicative senescence, the CDK1 and CDK2 protein expression was significantly increased 1:3 ± 0:2-fold and 1:2 ± 0:1-fold compared to young TMSCs, respectively. There was no significant change in cyclin B1 and cyclin E2 expression between the two groups in protein expression. Between the CDK1 and CDK2 proteins, CDK1 was selected as a strong senescence marker candidate for further analysis because it presented higher level of significance. To investigate the efficacy of CDK1 to cell proliferation, the real-time proliferation rates of the old TMSCs and CDK1-inhibited/transfected TMSCs were calculated. As shown in (Figures 6(a)-6(c)), it was confirmed that cell proliferation was significantly increased compared to the old TMSC control group in the CDK1/2-inhibited group (p < 0:01). In addition, the group treated with DMSO used as a solvent showed a similar growth rate trend as the old TMSC control group. It was indicated that CDK1/2 affects the proliferation rate in old TMSC. Additionally, we performed the transfection of CDK1 siRNA to investigate whether CDK1 has its own efficacy (Figures 6(d)-6(f)). The knockdown of CDK1 in old TMSCs significantly influenced cell proliferation compared to old TMSCs in doubling time (p < 0:001).

Discussion
As evidence continues to emerge regarding the importance of improving MSC proliferation and clinical therapy, MSC therapy is becoming an increasingly attractive alternative or additive for the treatment of various diseases [29]. Human tissuederived MSCs, including adipose-derived MSCs and bone marrow-derived MSCs, are ideal candidates for diverse regenerative medicine and tissue engineering strategies [30]. TMSCs have also been used for regeneration in various tissues, including bone [31], skeletal muscle [32], nerve [33], and parathyroid tissue [34]. To use MSCs for various clinical applications, replicative senescence is inevitable. However, many therapies involving MSCs often require ex vivo expansion approaches to generate the large number of cells required for the patients and to overcome the limitations of cell senescence [7]. Replicative senescence is defined as an irreversibly restricted proliferation due to telomere shortening in MSCs after a stereotypical number of cell divisions [35]. MSCs from elderly individuals recapitulate most parameters seen in senescent MSCs, including a flat, enlarged morphology, a large number of cells stained positive for SA-β-gal, and a lower proliferation rate. These characteristics have fueled the perception that replicative senescence in vitro may serve as a candidate model to unravel the molecular mechanisms that drive the process of body aging [36]. Replicative  Stem Cells International senescent MSCs dynamically change to senescence-related indicators, such as whole-map gene expression patterns and miRNA profiles. To identify biomarkers for senescent TMSCs, these changes should be considered therapeutic targets for MSC rejuvenation [37].
In this study, we established a senescent TMSC model in terms of morphological and biological characteristics in vitro. Senescent TMSCs were characterized by an enlarged and flattened cellular morphology by continuous subculture and increased SA-β-gal absorbance (Figures 1(a) and 1(b)). Along with the morphological changes, TMSC proliferation efficiency significantly decreased with replicative senescence and showed a prolonged G1 phase of the cell cycle and reduced S phase (Figures 1(c) and 1(d)). Moreover, our initial observations did not show changes in the hematopoietic and primitive markers between young and old TMSCs (Figure 2). The ability to detect changes in the expression of stem cell surface markers with senescence implies that more precise criteria are needed to identify senescent cells [17,38].
We have recently reported the alteration of ITGA3, integrin α3 protein in the cell surface, with senescence through transcriptomic analysis from a previous study [11]. In the current study, we found that the percentage of G1 cells increased drastically during the senescence process, and this phenomenon has been studied as the "Hayflick phenomenon" in which cells lose their ability to proliferate [39]. Replicative senescence activates DNA damage responses and preferentially kills the stationary phase function of proteins and the majority of the proteins that fail to arrest growth in G0/G1 [14]. Thus, in this study, we aimed to determine the factors that delay the cell cycle and cell proliferation in TMSCs.
We confirmed the significantly changed genes of the two groups divided to determine the overall transcriptomic gene pool of TMSCs changed by replicative senescence (Figure 3, Table 1). Transcripts that are simultaneously involved in the cell cycle and cellular senescence were collected, and their classification was determined (Figure 3(a)). The expression

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Stem Cells International of 18 transcripts, except HIPK2, was increased by replicative senescence. HIPK2 acts as a transcription factor that increases the DNA binding affinity and represses transcription [25,30]; however, it was probably affected by the decrease in expression because the amount that could be used for DNA damage caused by the senescence of TMSCs was reduced. Furthermore, according to the MF of the GO term with transcripts whose expression has changed (Figures 3(d)-3(f ), Supplementary Table 2), DNA functions, including catalytic activity on DNA, DNA helicase activity, DNA replication origin binding, and DNA-dependent ATPase activity showed a significant difference in expression. Additionally, the cyclin-dependent protein kinase regulator GO term category was also found to be significant, and the greatest effect of senescence on MSC proliferation was investigated by cell cycle change due to DNA damage. The KEGG pathway also showed that cell cycle-related transcripts play an essential role in the MSC replicative senescence process (Figure 4 and Supplementary Figure 1).
A paradox in the cellular response to DNA damage is that a checkpoint is enforced by inhibiting CDK activity, whereas many studies report that CDK activity is needed in a DNA damage response for DNA replication, 11 Stem Cells International homologous recombination, and DNA repair [40]. Our study identified significant increases in the expression of CDK1 and CDK2 genes in senescent TMSCs, which are known as cell division cycle protein homologs that are highly conserved and are key players in cell cycle regulation [24,41]. While the remaining CDK activity sustains important cellular functions in cell cycle, it also poses a risk for genome stability [42,43]. If cells with damaged DNA progress, they need to be prevented from entering mitosis, which otherwise could result in chromosome missegregation and propagation of mutations [44]. Therefore, the activity of residual CDK present in senescent TMSCs can rather promote cellular senescence.
In the present study, the CDK family, which was not phosphorylated due to DNA damage from replicative senescence, could not bind to the cyclin partner, resulting in the increased percentage of cells in the G1 phase (Figure 1(d)). The increased G1 phase due to decreased CDK inhibited cell proliferation and eventually showed the "Hayflick phenomenon." Quantitative gene expression and protein were also significantly increased in old TMSCs (Figures 4 and 5). Furthermore, cell proliferation efficiency was improved when CDK1 was inhibited/knocked down in old TMSCs ( Figure 6). These findings suggest that a transcriptome-based approach could provide greater discriminating power for establishing new senescence biomarkers for MSCs studies and serve as a basis for regulating established CDK1-induced senescence mechanisms.
In contrast, TGFB2, which is a pleiotropic cytokine that regulates a myriad of cellular processes, was not changed in quantitative RT-PCR results. Mounting evidence shows that the effects of TGF-β on the proliferation and senescence of MSCs have varied in the literature [45,46]. The TGF-β of senescence-promoting activity in several MSC types, including bone marrow MSCs and endometrial MSCs, has been previously described [47,48]. In bone marrow MSCs, TGF-β reportedly increases the expression levels of p16lnk4a and 4-Hydroxynonenal subunits, SA-β gal activity, senescence markers, and the production of mitochondrial ROS [47]. However, other studies have suggested that TGF-β promotes the proliferation of MSCs or does not affect senescence [49]. These contradictory observations might be partly explained by the heterogeneity of MSCs, as human MSCs contain at least three types that possess differentiation capacities [5,38].
Our transcriptomic analysis of senescent TMSCs revealed a much larger database, suggesting factors that can be selected for old MSCs. Future studies should investigate the detailed molecular mechanism, including changes in the intracellular localization of CDK1. It is necessary to check whether these results have the same effect in MSCs derived from other tissues. Additionally, further studies are needed to develop high-quality stem cell therapeutics through overall screening and mechanism research for various candidate substances capable of selecting senescent cells. These analyses have led us to propose that genes associated with the cell cycle and cellular senescence process are differentially altered with the aging of TMSCs, and some of these molecules could be used as potential indicators for identifying stem cells with replicative senescence.

Conclusions
In this study, we investigated transcriptomic changes in replicative senescent TMSCs compared to early passage TMSCs (young). We identified meaningful genes (CDK1 and CDK2) that consequently increased the proliferation of TMSCs. The CDK family could be used as an effective indicator or marker to improve quality control during isolation and expansion of MSCs, thereby increasing the therapeutic efficacy of MSCbased regenerative therapy. However, further investigations are required to fully evaluate the effectiveness and detailed molecular mechanisms, including changes in the intracellular localization of the CDK family. Additionally, other molecules classified into different GO term categories and related regulatory mechanisms remain to be identified.

Data Availability
The data that support the findings of this study are available upon request from the corresponding author. The data are not publicly available due to privacy concerns or ethical reasons.